Polypeptides implicated in the expression of resistance to glycopeptides, in particular in gram-positive bacteria, nucleotide sequence coding for these polypeptides and use for diagnosis

ABSTRACT

The invention relates to a composition of polypeptides, characterized in that it contains at least one protein or part of a protein selected from the sequences of amino acids identified in the list of the sequences as SEQ ID NO 1 (VanH), SEQ ID NO 2 (VanA). SEQ ID NO 3 (VanX) or SEQ ID NO 19 (VanC), or any protein or part of a protein recognized by the antibodies directed against VanH, VanA, VanX or VanC, or any protein or part of a protein encoded in a sequence hybridizing with one of the nucleotide sequences identified in the list of the sequences as SEQ ID NO 8, SEQ ID NO 9 or SEQ ID NO 10 or with one of the following sequences V1 or V2 under stringent or only slightly stringent conditions: 
     
       
         
               
               
             
                   
                 V1 : GGX GAA GAT GGX TCX TTX CAA GGX 
               
                   
                            G   C     AG  C     G 
               
                   
                                          A 
               
                   
                 V2 : AAT ACX ATX CCX GGX TTT AC 
               
                   
                        C     T             C 
               
                   
                              C 
               
           
              
              
              
              
              
              
             
          
         
       
     
     The invention also relates to the nucleotide sequences coding for these polypeptides as well as their utilization for the diagnosis of resistance to the glycopeptides.

This Application is a Divisional of application Ser. No. 08/980,357,filed on Nov. 28, 1997, now allowed, which is a Divisional ofapplication Ser. No. 08/286,819 filed on Aug. 5, 1994, now U.S. Pat. No.5,871,910, which is a Continuation of application Ser. No. 08/174,682filed on Dec. 28, 1993, abandoned, which is a Continuation ofapplication Ser. No. 07/917,146 filed on Aug. 10, 1992, abandoned, whichis a National Stage Application of PCT/FR91/00855 Oct. 29, 1991,abandoned.

The invention relates to the polypeptides associated with the expressionof resistance to antibiotics of the glycopeptide family, in particularin Gram-positive bacteria, in particular in the family of theGram-positive cocci. The invention also relates to a nucleotide sequencecoding for these polypeptides. It also relates to the use of thesepolypeptides and their nucleotide sequence as agents for the in vitrodetection of resistance to glycopeptides. Among the Gram-positive cocci,the invention relates most particularly to the enterococci, thestreptococci and the staphylococci which are of particular importancefor the implementation of the invention.

The glycopeptides, which include vancomycin and teicoplanin areantibiotics which inhibit the synthesis of the bacterial cell wall.These antibiotics are very much used for the treatment of severeinfections due to Gram-positive cocci (enterococci, streptococci andstaphylococci), in particular in the light of allergy and resistance tothe penicillins. In spite of long clinical usage of vancomycin, thisantibiotic has remained active towards almost all of the strains up to1986, the date at which the first resistant strains were isolated. Sincethen, resistance to the glycopeptides has been detected by manymicrobiologists in Europe and in the United States, in particular instrains isolated from immunodepressive patients, making necessary asystematic evaluation of the sensitivity of the microbes in hospitalenvironments.

The activity of the glycopeptides depends on the formation of a complexbetween the antibiotic and the precursors of the peptidoglycan, morethan on the direct interaction with enzymes of cell wall metabolism. Inparticular, it has been observed that the glycopeptides bind to theterminal D-alanyl-D-alanine residues (D-ala-D-ala) of the precursors ofthe peptidoglycan.

The recent emergence of resistance to the glycopeptides, in particularin the enterococci, has led to certain results being obtained withregard to knowledge of the factors conferring this resistance.

For example it has been observed in a particular strain of enterococci,Enterococcus faecium BM4147, that the determinant of resistance to theglycopeptides is localized on a plasmid of 34 kb, the plasmid pIP816.This determinant has been cloned in E. coli (Brisson Noel et al., 1990,Antimicrob Agents Chemother 34, 924-927).

According to the results hitherto obtained, the resistance to theglycopeptides is associated with the production of a protein ofmolecular weight of about 40 kDa, the synthesis of this protein beinginduced by sub-inhibitory concentrations of certain glycopeptides suchas vancomycin.

By carrying out a more detailed study of the resistance of certainstrains of Gram-positive cocci towards glycopeptides, in particularvancomycin or teicoplanin, the inventors have observed that thisresistance might be linked to the expression of several proteins orpolypeptides encoded in sequences usually borne by plasmids in theresistant strains. The recent results obtained by the inventors alsomake it possible to distinguish the genes coding for two phenotypes ofresistance, on the one hand strains highly resistant to theglycopeptides, and, on the other, strains with a low level ofresistance.

By strain with a high level of resistance is meant a strain of bacteria,in particular a strain of Gram-positive cocci, for which the minimalinhibitory concentrations (MIC) of vancomycin and teichoplanin arehigher than 32 and 8 μg/ml, respectively. The MIC of vancomycin towardsstrains with low-level resistance are included between 16 and 32 μg/ml.These strains are apparently sensitive to teicoplanin.

The inventors have isolated and purified, among the components necessaryfor the expression of the resistance to the glycopeptides, a particularprotein designated VANA or VanA which exhibits a certain homology withD-alanine-D-alanine ligases. VanA is nonetheless functionally distinctfrom the ligases.

In principle, a gene sequence will be designated by “van . . . ” and anamino acid sequence by “Van . . . ”.

The invention relates to polypeptides or proteins implicated in theexpression of resistance to antibiotics of the glycopeptide family and,in particular, to vancomycin and/or teicoplanin as well as to thenucleotide sequences coding for such complexes.

The invention also relates to nucleotide probes which can be used forthe detection of resistance to the glycopeptides, in particular by meansof the polymerase chain reaction (PCR), or by tests involvingantibodies.

The invention relates to a composition of polypeptides, characterized inthat it contains at least one protein or part of a protein selected fromthe amino acid sequences identified in the list of the sequences as SEQID NO 2 (VanH), SEQ ID NO 4 (VanA), SEQ ID NO 6 (VanX) or SEQ ID NO 8(VanC), or any protein or part of a protein recognized by the antibodiesdirected against VanH, VanA, VanX or VanC, or any protein or part of aprotein encoded in a sequence hybridizing with one of the nucleotidesequences identified in the list of the sequences as SEQ ID NO 1, SEQ IDNO 3, SEQ ID NO 5 or SEQ ID NO 7 or with one of the following sequencesV1 (SEQ ID NO:9) or V2 (SEQ ID NO:10) under stringent or only slightlystringent conditions:

V1 : GGX GAA GAT GGX TCX TTX CAA GGX            G   C     AG  C     G                         A V2 : AAT ACX ATX CCX GGX TTT AC       C     T              C              C

A first particular composition according to the invention implicated inthe expression of the resistance to the glycopeptides is characterizedin that it comprises at least 3 proteins or any part of one or more ofthese proteins necessary to confer to Gram-positive bacteria theresistance to antibiotics of the glycopeptide family, in particular tovancomycin and/or teicoplanin or to promote this resistance, inparticular in strains of the family of the Gram-positive cocci, theseproteins or parts of proteins being

-   a) recognized by antibodies directed against one of the sequences    identified in the list of the sequences as SEQ ID NO 2, SEQ ID NO 4,    SEQ ID NO 6,-   b) or encoded in genes containing a sequence identified as SEQ ID NO    1, SEQ ID NO 3 or SEQ ID NO 5 or hybridizing with one of these    sequences or its complementary sequence or with the sequences V1    (SEQ ID NO:9) or V2, (SEQ ID NO:10) under stringent or only slightly    stringent conditions.

These sequences are also designated, respectively, by ORF3, ORF1containing the gene VanH, vanA (or ORF2); they characterize the proteinsresponsible for resistance as obtained from the strain Enterococcusfaecium BM4147 described by Leclerq et al (N. Engl. J. Med.319:157-161).

Another protein, VanC, (SEQ ID NO:8) related to the D-Ala-D-Ala ligasesbut of different specificity has been characterized in Enterococcusgallinarum BM4173; the vanC gene (SEQ ID NO:7) possesses domains havingsufficient homology with the vanA gene for probes corresponding todefined regions of vanA to make possible its detection.

E. gallinarum is a constitutive isolate resistant to low levels ofvancomycin (Dutka-Malen et al., Antimicrob. Agents Chemother 34 (1990b)1875-1879).

By the expression “polypeptides” is meant any sequence of amino acidsconstituting proteins or being of a size less than that of a protein.

The stringent conditions mentioned above are defined according to theusual conditions pertaining to the hybridization of nucleotidesequences. As an example, in the case of the sequences which hybridizewith the sequence of the vanA gene (SEQ ID NO 1) it will be possible toapply the following conditions:

-   -   for hybridization under conditions of high stringency:        -   a reaction temperature of 65° C. overnight in a solution            containing 0.1% SDS, 0.7% skimmed milk powder, 6×SSC            (1×SSC=0.15 M NaCl and 0.015 M sodium citrate at pH=7.0)        -   washes at 65° C. in 2×SSC=0.1% SDS;    -   for hybridization under slightly stringent conditions, the        hybridization temperature is 60° C. overnight and the        temperature of the washings is 45° C.

The expression of resistance to glycopeptides may be expressed by thepersistence of an infection due to microbes usually sensitive to theglycopeptides.

A polypeptide or a protein is necessary for the expression of resistanceto the glycopeptides, if its absence makes the strain which containsthis polypeptide or this protein more sensitive to the glycopeptides andif this polypeptide or protein is not present in sensitive strains.

Different levels of resistance to the glycopeptides exist in the strainsof Gram-positive cocci, in particular.

According to a preferred embodiment of the invention, the polypeptidesincluded in the composition defined above correspond to the combinationof the proteins identified in the list of the sequences as SEQ ID NO 2(VanH), SEQ ID NO 4 (VanA), SEQ ID NO 6 (VanX).

The inventors have thus observed that the expression of resistance tothe glycopeptides in Gram-positive bacteria requires the expression ofat least three proteins or of polypeptides derived from these proteins.

According to a first particular embodiment of the invention, thepolypeptides of the composition are also characterized in that the aminoacid sequences necessary for the expression of resistance to antibioticsof the glycopeptide family are under the control of regulatory elements,in particular of the proteins corresponding to the sequences designatedby SEQ ID NO 12 and SEQ ID NO 14 in the list of the sequences, and whichcorrespond to a regulatory sequence R and to a sensor sequence S,respectively.

VanS and VanR constitute a two-component regulatory system, VanR beingan activator of transcription and VanS stimulating the transcriptiondependent on VanR. VanS is capable of modulating the level ofphosphorylation of VanR in response to the vancomycin present in theexternal medium and is thus involved in the control of the transcriptionof the genes for resistance to vancomycin.

These regulatory sequences are in particular capable of increasing thelevel of resistance, to the extent to which they promote the expressionof the proteins responsible for resistance comprised in the polypeptidesof the invention.

According to another advantageous embodiment of the invention, thepolypeptides of the above composition are encoded in the sequence SEQ IDNO 15 identified in the list of the sequences, which represents thesequence coding for the 5 proteins previously described.

Another sequence according to the invention is designated by SEQ ID NO16 which contains the sequence SEQ ID NO 15 as well as a sequenceupstream from SEQ ID NO 15 coding for a transposase (encoded in the (−)strand of the sequence, and a sequence downstream from SEQ ID NO 6corresponding to the genes vanY and vanZ and at each end reverserepeated sequences of 38 bp. SEQ ID NO 16 constitutes a transposon, thegenes of which are implicated at different levels in the establishmentof resistance to the glycopeptides.

The invention also relates to the purified proteins belonging to thecomposition and to the polypeptides described previously. In particular,the invention relates to the purified protein VanA, characterized inthat it corresponds to the amino acid sequence SEQ ID NO 4 in the listof the sequences or a protein VanC, encoded in a gene capable ofhybridizing with the vanA gene.

The protein VanA contains 343 amino acids and has a calculated molecularmass of 37400 Da. The protein VanC contains 343 amino acids and has acalculated molecular mass of 37504 Da.

Other interesting proteins in the framework of the invention correspondto the sequences identified as SEQ ID NO 2 (VanH), SEQ ID NO 6 (VanX),SEQ ID NO 12 (VanR), SEQ ID NO 14 (VanS) in the list of the sequences.

The sequence identified by the abbreviation SEQ ID NO 1 contains theprotein VanH encoded in the gene vanH, this protein contains 322 aminoacids and begins with a methionine. This protein is an enzyme implicatedin the synthesis of the peptidoglycan and has a molecular mass of 35,754kDA. VanH exhibits some similarities to dehydrogenases which catalyzethe NAD⁺-dependent oxidation of 2-hydroxy-carboxylic acids to form thecorresponding 2-keto-carboxylic acids. In fact, the VanH protein mightuse NADP⁺ rather than NAD⁺. The VanH protein also contains severalresidues of reactive sites which probably participate directly in thebinding of the substrate and in catalysis. VanH might be implicated inthe synthesis of a substrate of the ligase VanA. This substrate of VanAmight be a D-α-hydroxy-carboxylic acid, which might be condensed by VanAwith D-alanine in the place of a D-amino acid, which might affect thebinding of the precursor of the peptidoglycan with vancomycin, as aresult of the loss of a hydrogen bond because one of the hydrogen bondsformed between vancomycin and N-acetyl-D-Ala-D-Ala occurs with the NHgroup of the terminal D-alanine residue. Let it be recalled that “Ala”is the abbreviation for “alanine”.

The inventors have been able to detect some interactions between theproteins VanA and VanH and have in particular been able to describe thefollowing: the nature of the VanA protein (D-alanine: D-alanine ligasewith reduced specificity for its substrate) which has made possibleresistance to glycopeptides, implies the biosynthesis by VanA of a novelcompound different from D-Ala-D-Ala, a peptide which may be incorporatedinto the peptidoglycans but which is not recognized by vancomycin. Inparticular the observation of similarities between the product of thevanH gene and the D-specific α-keto-acid reductases has made it possibleto determine that this compound cannot be a D-amino acid but is a Dhydroxy acid, which when it is bound to D-alanine by VanH, can generatethe novel depsipeptide precursor of the peptidoglycan.

The invention also relates to any combination of these differentproteins in a resistance complex, as well as to hybrid proteinscomprising one or several of the above proteins, or part of theseproteins, in combination with a defined amino acid sequence.

Also included in the framework of the invention are nucleotide sequencescoding for one of the amino acid sequences described above.

A particular sequence is the nucleotide sequence of about 7.3 kb,corresponding to the HindIII-EcoRI restriction fragment, such as thatobtained starting from the plasmid pIP816 described in the publicationof Leclerq et al—1988, cited above.

This sequence of 7.3 kb comprises the nucleotide sequence coding for the3 resistance proteins and the 2 regulatory proteins referred to above.This coding sequence is included in an internal BglII-XbaI fragment. Italso comprises a part of the sequences coding for the transposase andthe resolvase.

The invention also relates to any nucleotide fragment comprising theabove-mentioned restriction fragment as well as any part of theHindIII-EcoRI fragment, in particular the EcoRI-XbaI fragment of about3.4 kb coding for the 3 resistance proteins or the EcoRV-SacII fragmentof about 1.7 kb coding for VanA or also HindIII-EcoRI fragment of about3.3 kb coding for the 2 regulatory proteins VanR and VanS.

Another definition of a nucleotide sequence of the invention correspondsto a nucleotide fragment containing the following restriction sites inthe following order, such as obtained starting from pIP816 mentionedabove:

-   HindIII, BglII, BglII, EcoRI, BamHI, XbaI, EcoRI.

Another nucleotide sequence according to the invention is characterizedin that it corresponds to a sequence selected from the sequencesidentified as SEQ ID NO 15, SEQ ID NO 17, or SEQ ID NO 16, or in that itincludes this sequence or any part of this sequence, or also anysequence or part of the sequence of the complementary DNA or anysequence of RNA corresponding to one of these DNAs, capable,

-   -   either of constituting a hybridization probe for the detection        of resistance to antibiotics of the glycopeptide family, in        particular to vancomycin and/or teicoplanin in particular in        strains of the family of the Gram-positive cocci,    -   or of coding for a sequence necessary or associated with the        expression of resistance to antibiotics of the glycopeptide        family, in particular to vancomycin and/or teicoplanin., in        particular in strains of the family of the Gram-positive cocci.

The sequence SEQ ID NO 17 codes for the 3 resistance proteins VanH, VanAand VanX.

The sequence SEQ ID NO 16 includes a transposon shown in FIG. 7 a; thistransposon contains the genes necessary for the expression of resistanceto the glycopeptides as well as the genes associated with thisresistance implicated, for example, in the regulation of the expressionof the genes necessary to produce the resistance phenotype or implicatedin the amount of resistance polypeptide produced.

A specific sequence corresponding to the above definition is one of thefollowing sequences:

  (SEQ ID NO:9) V1 : GGX GAA GAT GGX TCX TTX CAA GGX           G   C     AG  C     G or                       A   (SEQ IDNO:10) V2 : AAT ACX ATX CCX GGX TTT AC        C     T             T             C

V1 and V2 make possible the constitution of probes, if necessary, incombination with other nucleotides, depending on the degree ofspecificity desired in order to detect vanA and vanC and may also beused as primers in polymerase chain reactions.

Other preferred nucleotide sequences are the sequences SEQ ID NO 1, SEQID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID 18 (transposase), SEQ ID NO 20(resolvase), SEQ ID NO 22 (vanY), SEQ ID NO 24 (vanZ), SEQ ID NO 11(vanR), SEQ ID NO 13 (vanS) or a variant of one of these sequencesprovided that it codes for a protein having immunological and/orfunctional properties similar to those of the proteins encoded in thesequences SEQ ID NO 1 (vanA), SEQ ID NO 3 (vanH), SEQ ID NO 5 (vanX), orSEQ ID NO 7 (vanC), SEQ ID NO 18 (transposase), SEQ ID NO 20(resolvase), SEQ ID NO 22 (vanY), SEQ ID NO 24 (vanZ), SEQ ID NO 11(vanR), SEQ ID NO 13 (vanS) or in that it makes possible the detectionof strains resistant to antibiotics of the glycopeptide family.

Variants include all of the fragments of the sequences having thefollowing properties.

These sequences code for the resistance proteins VanH, VanA and VanX.

The nucleotide sequence designated by SEQ ID NO 1 corresponds to a DNAfragment of 1029 bp situated between the ATG codon at position 377 andthe TGA codon at position 1406 on the plasmid pAT214 (FIG. 6).

The invention also relates to a nucleotide sequence coding for thesequence SEQ ID NO 15 corresponding to the sequence coding for the 5proteins (2 regulatory proteins and 3 resistance proteins), and alsocomprising the flanking sequences associated with these codingsequences, or comprising this sequence.

Also included in the framework of the invention is a sequence modifiedwith respect to SEQ ID NO 15, characterized in that it lacks theflanking sequences. These flanking sequences are the sequences shown inthe following pages and defined as follows:

-   -   sequence upstream from the sequence coding for R: between the        bases 1 and 1476 of the sequence shown in FIG. 5,    -   sequence between the sequence coding for the sensor protein S        and ORF1: between the bases 3347 and 3500 of the sequence shown        in FIG. 5,    -   sequence downstream from the sequence coding for ORF3: between        the bases 6168 and 7227 of the sequence shown in FIG. 5.

The sequence designated by SEQ ID NO 15 is also characterized by thefragment bearing the restriction sites in the following order:

-   -   BglIII-EcoRI-BamHI-EcoRI

The location of the regulatory proteins and the resistance proteins isshown in FIG. 3.

The inventors have identified upstream and downstream from the genesvanR, vanS, vanH, vanA and vanX, which are necessary for or associatedwith the expression of resistance to glycopeptides at a given level,genes coding for a transposase and a resolvase (upstream from the grouppreviously mentioned) and genes vanY and vanZ, downstream from thisgroup. The genes for the transposase and resolvase might be implicatedin transposition functions and the vanY gene coding for a D,D-carboxypeptidase might be implicated in the metabolism of the peptidoglycan,and might contribute to resistance to the glycopeptides in E. faeciumBM4147 even though vanR, vanS, vanH, vanA and vanX borne by a plasmid ina high number of copies, alone confer a high level of resistance.

Let it be noted that the sequence coding for the transposase (SEQ IDNO:18) is located on the (−) strand of the sequence ID NO 16 which codesfor vanR, vanS, vanH, vanA, vanX, vanY, vanZ and the resolvase.

The invention relates not only to the DNA sequences identified in thelist of the sequences but also to the complementary DNA sequences andthe corresponding RNA sequences. The invention concerns in additionsequences which are equivalent to the former, either in terms ofexpression of proteins, polypeptides or their fragments described above,or in terms of the capacity to detect, for example by chainpolymerization procedures, strains of Gram-positive bacteria exhibitingresistance to antibiotics of the glycopeptide family such as vancomycinor teicoplanin.

Recombinant sequences characterized in that they comprise one of theabove nucleotide sequences also form part of the invention.

The invention also relates to a recombinant vector characterized in thatit includes one of the above nucleotide sequences at a site inessentialfor its replication, under the control of regulatory elements likely tobe implemented in the expression of the resistance to antibiotics of theglycopeptide family, in particular to vancomycin or teicoplanin, in adefined host.

Particularly advantageous recombinant vectors for the implementation ofthe invention are the following vectors: pAT214 containing theEcoRV-SacII fragment of 1761 bp containing a nucleotide sequence codingfor the VanA protein; in these vectors the sequences of the inventionare advantageously placed under the control of promoters such as the lacpromoter.

The invention also relates to a recombinant cell host containing anucleotide sequence such as that previously described or a vector suchas that described above under conditions which make possible theexpression of resistance to antibiotics of the glycopeptide family, inparticular resistance to vancomycin and/or this host being for exampleselected from the bacteria, in particular the Gram-positive cocci.

In certain applications it is also possible to use yeasts, fungi, insector mammalian cells.

The invention also relates to a nucleotide probe characterized in thatit is capable of hybridizing with a sequence previously described, thisprobe being labelled if necessary. These probes may or may not bespecific for the proteins of resistance to glycopeptides.

Labels which can be used for the requirements of the invention are theknown radioactive labels as well as other labels such as enzymaticlabels or chemoluminescent labels.

Probes thus labelled may be used in hybridization tests in order todetect resistance to glycopeptides in Gram-positive bacteria. In thiscase, conditions of low stringency will be used.

Nucleotide probes according to the invention may be characterized inthat they are specific in Gram-positive bacteria for the sequencescoding for a resistance protein to the glycopeptides, in particular tovancomycin and/or teicoplanin these probes being in addition universalamong these sequences.

By these specific probes is meant any oligonucleotide hybridizing with anucleotide sequence coding for one of the proteins according to theinvention, such as described in the preceding pages, and not exhibitinga cross hybridization reaction or amplification reaction (PCR) withsequences present in all of the sensitive strains.

The universal character of the oligonucleotide which can be used in PCRis defined by their capacity to promote specifically the amplificationof a nucleotide sequence implicated in resistance in any one strain ofGram-positive bacteria, resistant to the antibiotics of the glycopeptidefamily.

The size of the nucleotide probes according to the invention may varydepending on the use desired. For the oligonucleotides which are used inPCR, recourse will be had to fragments of a length which is usual inthis procedure. In order to construct probes, it is possible to take anypart of the sequences of the invention, for example probe fragments of200 nucleotides.

According to a particular embodiment of the invention, a nucleotideprobe is selected for its specificity towards a nucleotide sequencecoding for a protein necessary for the expression in Gram-positivebacteria of a high level of resistance to antibiotics of theglycopeptide family, in particular to vancomycin and teicoplanin.

As examples, useful probes may be selected from the intragenic part ofthe vanA gene.

Other useful probes for carrying out the invention are characterized bytheir universal character, according to the preceding definition, butare not specific for the resistance genes. They may also be used asprimers in PCR, and are for example:

  (SEQ ID NO:9) V1 : GGX GAA GAT GGX TCX TTX CAA GGX           G   C     AG  C     G                          A   (SEQ IDNO:10) V2 : AAT ACX ATX CCX GGX TTT AC        C     T             C             C

V1 and V2 hybridize with vanA and vanC and are capable of leading to thedetection of proteins associated with resistance to glycopeptides inother micro-organisms.

Other particular probes of the invention have the specific character ofa nucleotide sequence coding for a protein necessary for the expressionin Gram-positive bacteria of a low level of resistance to antibiotics ofthe glycopeptide family, in particular to vancomycin in Gram-positivebacteria.

It should also be mentioned that oligonucleotide probes which might bederived from the sequence of the vanA gene coding for the VanA proteinmay be used indiscriminately to detect high-level or low-levelresistance.

In a particularly preferred manner, a probe of the invention ischaracterized in that it hybridizes with a chromosomal ornon-chromosomal nucleotide sequence of a Gram-positive strain resistantto glycopeptides, in particular to vancomycin and/or teicoplanin, inparticular in that it hybridizes with a chromosomal or non-chromosomalnucleotide sequence of a strain of Gram-positive cocci, for example anenterococcal strain and preferably E. faecium 4147 or E. gallinarum.

In order to distinguish strains with a high level of resistance fromstrains with a low level of resistance it is possible to carry out ahybridization test using conditions of high stringency.

The oligonucleotides of the invention may be obtained from the sequencesof the invention by cutting with restriction enzymes, or by chemicalsynthesis according to the standard methods.

Furthermore, the invention relates to polyclonal or monoclonalantibodies, characterized in that they recognize the polypeptide(s)described above or an amino acid sequence described above.

These antibodies may be obtained according to standard methods forantibody production. In particular, in the case of the preparation ofmonoclonal antibodies, recourse will be had to the method of Köhler andMilstein according to which monoclonal antibodies are prepared by cellfusion between myeloma cells and mouse spleen cells previously immunizedwith a polypeptide or a composition according to the invention, inconformity with the standard procedure.

The antibodies of the invention can advantageously be used for thedetection of the presence of proteins characteristic of resistance tothe glycopeptides, in particular to vancomycin and teicoplanin.

Particularly useful antibodies are polyclonal or monoclonal antibodiesdirected against the protein VanA or VanC. Such antibodiesadvantageously make it possible to detect strains of bacteria, inparticular Gram-positive cocci, exhibiting high-level resistance to theantibiotics of the glycopeptide family. If necessary, a step entailinglysis of the cells of the sample undergoing detection is performed priorto the placing in contact of the sample with the antibodies.

In order to carry out this detection, recourse will advantageously behad to antibodies labelled for example with a radioactive substance orother type of label.

Hence, tests for the detection in Gram-positive bacteria of resistanceto the glycopeptides, in particular tests making use of the ELISAprocedures, are included in the framework of the invention.

A kit for the in vitro diagnosis of the presence of Gram-positivestrains, resistant to the glycopeptides, in particular to vancomycinand/or teicoplanin, these strains belonging in particular to theGram-positive cocci for example enterococci, for example E. faecium orE. gallinarum is characterized in that it comprises:

-   -   antibodies corresponding to the above definition, labelled if        necessary,    -   a reagent for the detection of an immunological reaction of the        antigen-antibody type,    -   if necessary, reagents to effect the lysis of the cells of the        sample to be tested.

Furthermore, the agents developed by the inventors offer the very usefuladvantage of being suitable for the development of a rapid and reliabletest or kit for the detection of Gram-positive strains resistant to theglycopeptides by means of the polymerase chain reaction (PCR). Such atest makes it possible to improve the sensitivity of the existing testswhich remain rather unreliable and, in certain cases, may make possiblethe detection of all of the representatives of the family of the genescoding for resistance proteins to the glycopeptides in Gram-positivebacteria.

The carrying out of a test by means of the method of amplification ofthe genes of these proteins is done by the PCR procedure or by the RPCRprocedure (RPCR: abbreviation for reverse polymerase chain reaction).

The RPCR technique makes possible the amplification of the NH₂ and COOHterminal regions of the genes it is desired to detect.

Some specific primers make it possible to amplify the genes of thestrains with low-level resistance. These primers are selected, forexample, from the sequence coding for the resistance protein VanA.

As examples, the following sequences can be used as primers for thepreparation of probes for the detection of an amplification by means ofthe PCR or RPCR method.

  (SEQ ID NO:9) V1 : GGX GAA GAT GGX TCX TTX CAA GGX           G   C     AG  C     G                          A   (SEQ IDNO:10) V2 : AAT ACX ATX CCX GGX TTT AC        C     T             C             C

X represents one of the bases A, T, C or G or also corresponds in allcases to inosine.

Naturally, the invention relates to the complementary probes of theoligonucleotides previously described as well as possibly to the RNAprobes which correspond to them.

A kit for the in vitro diagnosis of the presence of strains ofGram-positive bacteria resistant to the glycopeptides, in particularresistant to vancomycin and/or teicoplanin these strains belonging inparticular to the Gram-positive cocci, in particular that they arestrains of enterococci, for example E. faecium or E. gallinarum, ischaracterized in that it contains:

-   -   a nucleotide probe complying with the above specifications and        if necessary,    -   oligonucleoside triphosphates in an amount sufficient to make        possible the amplification of the desired sequence,    -   a hybridization buffer,    -   a DNA polymerization agent.

The invention also relates to a procedure for the in vitro detection ofthe presence of Gram-positive strains resistant to the glycopeptides, inparticular to vancomycin and/or teicoplanin these strains belonging inparticular to the family of the Gram-positive cocci, in particular inthat they are strains of enterococci, for example E. faecium or E.gallinarum, characterized in that it comprises:

-   a) the placing of a biological sample likely to contain the    resistant strains in contact with a primer constituted by a    nucleotide sequence described above, or any part of a sequence    previously described, capable of hybridizing with a desired    nucleotide sequence necessary for the expression of resistance to    the glycopeptides, this sequence being used as matrix in the    presence of the 4 different nucleoside triphosphates and a    polymerization agent under conditions of hybridization such that for    each nucleotide sequence which has hybridized with a primer, an    elongation product of each primer complementary to the matrix is    synthesized,-   b) the separation of the matrix from the elongation product    obtained, this latter then also being capable of behaving as a    matrix,-   c) the repetition of step a) so as to produce a detectable amount of    the desired nucleotide sequences,-   d) the detection of the product of amplification of the nucleotide    sequences.

The detection of the elongation products of the desired sequence may becarried out by a probe identical with the primers used to carry out thePCR or RPCR procedure, or also by a probe different from these primers,this probe being labelled if necessary.

Details relating to the implementation of the PCR procedures may beobtained from the patent applications EP 0229701 and EP 0200362.

Other advantages and characteristics of the invention will becomeapparent in the examples which follow and from the figures.

FIGURES

FIG. 1 electrophoresis on SDS-polyacrylamide gel (SDS-PAGE) of theproteins of the membrane fractions line 1 and line 4, molecular weightstandards; line 2, E. faecium BM4147 placed in culture in the absence ofvancomycin; line 3, BM4147 placed in culture in the presence of 10 μg/mlof vancomycin. The head of the arrow indicates the position of the VanAprotein.

FIG. 2:

FIG. 2A Restriction maps of the inserts of the plasmids pAT213 andpAT214. The vector and the DNA insert are distinguished by light anddark segments, respectively. The open arrow represents the vanA gene.

FIG. 2B Strategy for the nucleotide sequencing of the insert of 1761 bpin the plasmid pAT214. The arrows indicate the direction and extent ofthe sequencing reactions by the dideoxy method. The syntheticoligonucleotide primer (5′ ATGCTCCTGTCTCCTTTC 3′ OH) (SEQ ID NO:27) iscomplementary to the sequence between the positions 361 and 378. Onlythe pertinent restriction sites are given.

FIG. 3: position of the sequences R, S, ORF1, ORF2, ORF3.

FIG. 4: representation of SEQ ID NO 15.

FIG. 5: representation of SEQ ID NO 15 and the corresponding protein.(SEQ ID NOS:27, 28 and 29).

FIG. 6: sequence of the vanA gene and the corresponding protein.

FIG. 7:

(A): Localization of the genes vanR, vanS, vanes, vanA, vanX, vanY, vanZof the gene for the transposase and of the gene for the resolvase aswell as the repeated reverse terminal sequences of 38 bp at the end ofthe transposon.

Mapping of the plasmids. (B) Polylinker pAT29 and derivativesconstructed in this study. The arrow labelled P2 indicates the positionand orientation of the P2 promoter of aphA-3 (Caillaud et al., 1987,Mol. Gen. Genet. 207:509-513). (C) Insert pAT80. The white rectanglesindicate the DNA of pAT29 but they are not shown to scale. Therectangles terminating in an arrow indicate the coding sequences. Thearrows shown in vertical and horizontal full lines indicate the positionand orientation, respectively, of the apha-1 gene in the derivatives ofpAT80. Restriction sites: Ac, AccI; B, BamHI; Bg, BglII; Bs, BssHII; E,EcoRI; H, HindIII; Hc, HincII; K, KpnI; P, PstI; S, SmaI; SI, SacI, SII,SacII; Sa, SalI; Sp, SphI; Xb, XbaI. (D) Inserts in pAT86, pAT87, pAT88and pAT89. The inserts are shown by full lines and the correspondingvectors are indicated in parentheses.

FIGS. 8 A-W: nucleotide sequence of the transposon shown in FIG. 7 andamino acid sequence of the corresponding proteins. The nucleotidesequence is shown for the (+) strand and for the (−) strand(corresponding to the complementary sequence of the (+) strand for thepositions 1 to 3189) on which the coding sequence of the transposase islocated.

FIGS. 9 A-C: Nucleotide sequence of the SacI-PstI fragment of 1347 bp ofthe plasmid pAT216 containing the vanC gene. The numbering starts at thefirst base G of the SacI restriction site. The potential RBS sequenceupstream from the initiation codon ATG of translation at position 215 isunderlined. The STOP codon (TGA) is indicated by *. The region codingfor the vanC and the deduced amino acid sequence are indicated in boldcharacters. Sequential overlapping clones were generated by restrictionfragments of subcloning of pAT216 in the bacteriophage M13 mp10(Amersham, England). The universal primer (New England Biolabs BeverlyMass.) was used to sequence the insert in the recombinant phages. Thesequencing was performed by the enzymatic dideoxy nucleotide method(Sanger et al., 1977 PNAS 74: 5463-5467) by using the T7 DNA polymerase(Sequenase US B CORP, Cleveland, Ohio) and [α-³⁵S] dATP (Amersham,England). The reaction products were loaded onto 6% denaturingpolyacrylamide gels.

FIG. 10: alignment of the amino acid sequence of VanC (SEQ ID NO:8),VanA (SEQ ID NO:4), Dd1A (SEQ ID NO:32) and Dd1B (SEQ ID NO:33). Theidentical (I) amino acids and the conservative (C) substitutions in the4 sequences are indicated in the alignment. In order to classify theconservative substitutions, the amino acids were grouped as follows: RK,LFPMV1 (SEQ ID NO:34), STQNC (SEQ ID NO:35), AGW, H, ED AND Y. Theregions of high homology corresponding to the domains 1, 2, 3 and 4 areunderlined. The sequences corresponding to the peptides of 1 and 2 areindicated by the arrows.

FIG. 11: description of the oligonucleotides V1 (SEQ ID NO:9) and V2(SEQ ID NO:10) (A): Amino acid sequence of the peptides 1 (SEQ ID NO:36)and 2 (SEQ ID NO:37) of VanA and of the D-Ala-D-Ala ligases (SEQ IDNOS:36-39). The number of amino acids between the N-terminus and peptide1, between the peptides 1 and 2 and the peptide 2 and the C-terminus isindicated. The identical amino acids between at least 2 of the 3sequences are indicated in bold characters.

FIG. 11B: Target peptides (SEQ ID NO:36-39) and deduced nucleotidesequence. X represents any base of the DNA. Peptide 2 in Dd1B (SEQ IDNO:39) differs from the target peptide at 2 positions (*).

FIG. 11C: Nucleotide sequence of V1 (SEQ ID NO:9) and V2 (SEQ ID NO:10). Alternate nucleotides and deoxyinosine (I) which may correspond to anybase in the DNA, were used at the positions at which the nucleotidesequences coding for the target peptides vary. The arrows indicate thedirection of DNA synthesis. The oligonucleotides were synthesized by themethoxy-phosphoramidite method with a Biosystem DNA 380B machine(Applied Biosystem, Foster City, Calif.). The DNA was isolated frombacterial lysates by extraction with hexadecyl trimethyl ammoniumbromide (Inst. biotechnologies, Inc., New Haven, Colo.) (Le Bouguénec etal., 1990, J. Bacteriol. 172:727-734) and used as matrix for theamplification by means of PCR with a controlled heating system“Intelligent Heating Block” IBH101 (Hybarid Ltd., GB) according to thedescription of Mabilat et al. (1990, Plasmid 23:27-34). Theamplification products were revealed by electrophoresis on a 0.8% gel,after staining with ethidium bromide.

FIG. 12: Inactivation by insertion of vanC. The vanC gene is shown by anopen arrow and the internal EcoRI-HincII fragment of 690 bp is hatched.The DNA of pAT114 is shown by a thin line; the chromosomal DNA of PM4174by a thick line; the arrows indicate the genes for resistance to theantibiotics: aphA-3 is the gene coding for the 3′-aminoglycosidephosphotransferase; erm is the gene coding for the ER^(R) methyltransferase.

FIG. 12A: The plasmid pAT217 was constructed by ligation of theEcoRI-HincII fragment of pAT216 to the suicide vector pAT114 (Trieu-Cuotet al., 1991, Gene 106:21-27), digested with EcoRI and SmaI.

FIG. 12B: vanC region of the chromosomal DNA of BM4174.

FIG. 12C: vanC region after integration of pAT217.

FIG. 13: Southern blot analysis of the integration of pAT217 into thevanC gene of BM4174.

(left hand side): Total DNA of BM4175 (line 2) and BM4174 (line 3)digested with EcoRI and resolved by means of electrophoresis on a 1%agarose gel. The DNA of the bacteriophage lambda digested with PstI wasused as molecular mass standard (line 1). The DNA was transferred undervacuum to a Nytran membrane (Schleicher and Schül, Germany) by using aTrans-Vac TE80 apparatus (Höfer Scientific Instruments, San Francisco,Calif.) and bound to the membrane through the intermediary of UV light.The hybridization was carried out with the probe C (Middle) or the probeaphA-3 specific for pAT114 (Lambert et al., 1985, Annales de l'InstitutPasteur/Microbiol. 136(b): 135-150).

(right hand side): the probes were labelled with ³²P by nicktranslation. The molecular masses (kb) are indicated.

FIG. 14: alignment of the deduced amino acid sequences of VanS derivedfrom E. faecium BM147 (SEQ ID NO:40) and of PhoR (SEQ ID NO:41) and EnvZ(SEQ ID NO:42) from E. coli. The numbers on the left refer to theposition of the first amino acid in the alignment. The numbers on theright refer to the position of the last amino acid of the correspondingline. The identical amino acids are placed in boxes. The dotted linesindicate gaps introduced in order to optimize their similarity. Thedashes indicate the positions of the amino acid residues conserved inother HPK. The histidine residues in bold characters in section 1 arepotential sites of auto-phosphorylation.

FIG. 15: alignment of the deduced amino acid sequences of VanR from E.faecium BM4147(SEQ ID NO:43) , OmpR (SEQ ID NO:44) and PhoB (SEQ IDNO:45) from E. coli as that of CheY from Salmonella typhimurium (SEQ IDNO:46). The numbers on the right indicate the position of the last aminoacid of the corresponding line. The identical amino acids are placed inboxes. The dotted lines indicate the gaps introduced in order tooptimize the homologies. The residues in bold characters correspond tothe amino acids strongly conserved in the effector domains of other RR.The aspartic acid residue 57 of CheY is phosphorylated by the HPKassociated with CheA.

I—Identification of VanA

Materials and Methods for the Identification and Characterization of theVanA Gene

Bacterial Strains and Plasmids

The origin of the plasmids used is given in the table below.

Strain or plasmid Source or reference Escherichia coli JM83 Messing(1979) AR1062 Rambach and Hogness (1977) JM103 Hannshan (1983) ST640Lugtenberg and van Schijndel van-Dam (1973) Enterococcus faecium BM4147Leclercq et al (1988) Plasmid pUC18 Norrander et al (1983) pAT213Brisson-Noel et al (1990) pAT214 Described in this text

Preparation of the Enterococcal Membranes

Enterococcus faecium BM4147 was cultivated in 500 ml of heart-brainbroth (BHI broth medium) until the optical density (OD₆₀₀) reached 0.7.Induction was effected with 10 μg/ml of vancomycin (Eli LillyIndianapolis Ind.). The subsequent steps were performed at 4° C. Thecells were recovered by centrifugation for 10 minutes at 6000 g, washedwith a TE buffer (0.01 M TRIS-HCl, 0.002 M EDTA, pH 7.0) and lysed byglass beads (100 μm in diameter) in a Braun apparatus for 2 minutes. Thecell debris were separated by centrifugation for 10 minutes at 6000 g.The membranes were collected by centrifugation for 1 hour at 65000 g andresuspended in 0.5 ml of TE buffer.

Preparation of the Minicells

Plasmids were introduced by transformation into the strain E. coliAR1062 prepared in the form of bacterial vesicles. The bacterialvesicles were recovered on sucrose gradients and the proteins werelabelled with 50 μCi of [³⁵S]-L-methionine (Amersham, Great Britain)according to the method of Rambach and Hogness (1977, P.N.A.S. USA, 74;5041-5045).

Preparation of the Membrane Fractions and the Cytoplasmic Fractions ofE. coli

E. coli JM83 and strains derived from it were placed in culture in BHImedium until an optical density (OD₆₀₀) of 0.7 was attained, washed andsuspended in a TE buffer. The cell suspension was treated by sonication(ultrasound) for 20 seconds at doses of 50 W in a cell fragmentationapparatus in a Branson B7 sonication apparatus and the intact cells wereremoved by centrifugation for 10 minutes at 6000 g. The supernatant wasfractionated into membrane and cytoplasmic fractions by means ofcentrifugation for 1 hour at 100,000 g.

Electrophoresis on SDS-polyacrylamide Gel (SDS-PAGE)

The proteins from the bacterial fractions were separated by means ofSDS-PAGE on linear gradients of polyacrylamide gels (7.5%-15%) (Laemmli1970, Nature 227: 680-685). The electrophoresis was carried out for 1hour at 200 V, then for 3 hours at 350 V. The gels were stained withCoomassie blue. The proteins of the extracts were separated on 10%polyacrylamide gels and visualized by means of autoradiography.

Purification of the Protein Band and Determination of the N-TerminalSequence

The proteins of the membrane fractions of an induced culture of E.faecium BM14147 were separated by means of SDS-PAGE. The gel waselectrotransferred for 1 hour at 200 mA to a polyvinylidene difluoridemembrane (Immobilon Transfer, Millipore) by using a transfer apparatus(Electrophoresis Unit LKB 2117 Multiphor II) in accordance with theinstructions of the manufacturer. The transferred proteins were stainedwith Ponceau red. The portion of membrane bearing the protein ofinterest was excised, centered on a Teflon filter and placed in thecartridge of a sequencer (Sequencer Applied Biosystems model 470A). Theprotein was sequenced by means of the automated Edman degradation (1967,Eur. J. Biochem. 1; 80-81).

Construction of Plasmids

The plasmid pAT213 (Brisson-Noel et al., 1990, Antimicrob. AgentsChemother., 34; 924-927) consists of a EcoRI fragment of DNA of 4.0 kbof the enterococcal plasmid pIP816 cloned at the EcoRI site of aGram-positive-Gram-negative shuttle vector pAT187 (Trieu-Cuot et al.,1987, FEMS Microbiol. Lett. 48; 289-294). In order to construct pAT214,the EcoRV-SacII DNA fragment of 1761 bp of pAT213 was purified, treatedwith the Klenow fragment of the DNA polymerase I of E. coli and ligatedto the DNA of pUC18 which had previously been digested with SmaI anddephosphorylated (FIG. 2). The cloning (Maniatis et al., 1982 ColdSpring Harbor Laboratory Press) was carried out with restrictionendonucleases (Boehringer Mannheim and Pharmacia), with the T4 DNAligase (Pharmacia) and alkaline phosphatase (Pharmacia) according to theinstructions of the manufacturer.

Subcloning in M13 and Nucleotide Sequence

The DNA restriction fragments were subcloned in the polylinker of thereplicative forms of the derivatives mp18 and mp19 of the bacteriophageM13 (Norrander et al., 1983, Gene 26; 101-106), obtained from PharmaciaP-L Biochemicals. E. coli JM103 was transfected with recombinant phagesand the single-stranded DNA was prepared. The nucleotide sequencing wascarried out by the enzymatic di-deoxy nucleotide method (Sanger et al.,1977, P.N.A.S. USA 74; 5463-5467) by using a T7 DNA polymerase(Sequenase, United States Biochemical Corporation, Cleveland, Ohio) and[α-³⁵S] dATP (Amersham, Great Britain). The reaction products wererevealed on 6% polyacrylamide gels containing a denaturing buffer.

Data-Processing Analysis and Data on the Sequence

The complete DNA sequence was assembled by using the computer programsDBCOMP and DBUTIL (Staden, 1980, Nucleic Acids Res 8; 3673-3694). Theprotein data bank PSEQIP of the Pasteur Institute was screened using analgorithm developed by Claverie (1984, Nucleic Acids Res 12; 397-407).The alignments between the pairs of amino acid sequences wereconstructed using the algorithm of Wilbur et al (1983, P.N.A.S. USA 80;726-730). The statistical significance of the homology was evaluatedwith the algorithm of Lipman and Pearson (1985, Science 227; 1435-1440).

For each comparison 20 amino acid sequences were used to calculate themean values and the standard deviations of the random results.

Genetic Complementation Tests

The plasmids were introduced by transformation into E. coli ST640, atemperature-sensitive mutant with an unmodified D-ala-D-ala ligase(Lugtenberg et al 1973, J. Bacteriol 110; 26-34). The transformants wereselected at 30° C. on plates containing 100 μg/ml of ampicillin and thepresence of the plasmid DNA of the expected size and the restrictionmaps were verified. Single colonies grown at 30° C. in BHI broth mediumcontaining ampicillin were placed on a BHI agar medium containing both100 μg/ml of ampicillin and 50 μM ofisopropyl-1-thio-β-D-galacto-pyranoside (IPTG) and the plates wereincubated at a permissive temperature of 30° C. and at a non-permissivetemperature of 42° C. The complementation test was considered to bepositive if the colonies were present after 18 hours of incubation at42° C.

Results

Identification of the VanA Protein and its N-Terminal Sequence

The membrane fractions of the E. faecium BM4147 cells placed in culture,on the one hand, under conditions of induction, and, on the other, inthe absence of induction, were analysed by means of SDS-PAGE. The soledifference which could be detected, related to the exposure tosub-inhibitory concentrations of vancomycin, was the markedintensification of a band which corresponded to a protein of anestimated molecular weight of about 40 kDa. In the induced cells and inthe non-induced cells, the protein band represents the same proteinbecause this band is absent from membranes of a derivative of BM4147which has lost the pIP816 plasmid. The inducible protein, designated asVanA, was purified after SDS-PAGE and automated Edman degradation wascarried out on a 50 pmol. sample. Nine amino acids of the N-terminalsequence of VanA were identified: Met Asn Arg Ile Lys Val Ala Ile Leu(SEQ ID NO:47).

Sub-Cloning of the VanA Gene

The insert of 4.0 kb of the plasmid pAT213 bears the determinant forresistance to the glycopeptides of E. faecium BM4147. Variousrestriction fragments of this insert were subcloned in pUC18 and therecombinant plasmids specific for vanA in E. coli were identified bySDS-PAGE analysis of the proteins of the cytoplasmic and membranefractions or of the extracts of the bacterial vesicles. This approachwas used since E. coli is intrinsically resistant to the glycopeptide.The EcoRV-SacII insert of the pAT214 plasmid (FIG. 2) codes for a uniquepolypeptide of 40 kDa which migrates together with VanA, derived fromthe membrane preparations of E. faecium BM4147.

Nucleotide Sequence of the Insert in pAT214 and Identification of theVanA Coding Sequence

The nucleotide sequence of the EcoRV-SacII insert of 1761 bp in pAT214was determined on both strands of the DNA according to the strategydescribed in FIG. 2. The location of the termination codons (TGA, TAA,TAG) in three reading frames on each DNA strand showed the presence of aunique open reading frame (ORF) which was sufficiently long to code forthe VanA protein. This reading frame ORF is located between the TAAcodon at position 281 and the TAG codon at position 1406. The amino acidsequence deduced for ORF was compared with that of the N-terminus ofVanA. The nine amino acids identified by protein sequencing are encodedin the nucleotide sequence beginning with the ATG (methionine) codon atposition 377 (FIG. 3). This codon for the initiation of translation ispreceded by a sequence (TGAAAGGAGA)(SEQ ID NO:48) characteristic of aribosomal binding site (RBS) in Gram-positive bacteria which iscomplementary to the 8 bases of the rRNA of the 16S subunit of Bacillussubtilis in its sequence (3′ OH UCUUUCCUCC 5′) (SEQ ID NO:49) (Moran etal., 1982, Mol. Gen. Genet. 186; 339-346). In this ORF, there is noother ATG or GTG initiation codon between the positions 281 and 377. Thesequence of 1029 bp which extends from the ATG codon at position 377 tothe TGA codon at position 1406 codes for a protein containing 343 aminoacid residues. The calculated molecular weight of this protein is 37400Da, which is in agreement with the estimation of 40 kDa obtained bySDS-PAGE analysis.

Homology of the Amino Acid Sequences of VanA and the D-ala-D-ala LigaseEnzymes

The screening of the protein data bank PSEQIP has shown the existence ofa sequence homology between VanA and the D-ala-D-ala ligases of E. coli(ECOALA, Robinson et al., 1986, J. Bacteriol. 167; 809-817) and ofSalmonella typhimurium (DALIG, Daub et al., 1988, Biochemistry 27;3701-3708). The calculated percentage of homology between pairs ofproteins was included between 28% and 36% for the identical amino acidsand between 48% and 55% by taking into consideration homologous aminoacids. VanA (SEQ ID NO:4) and DALIG are more closely related. Thestatistical significance of these similarities wa evaluated by aligningVANA and sequences containing the same composition of amino acids asDALIG or ECOALA (Lipman and Pearson, 1985, Science 227; 1435-1440).

Genetic Complementation Test for the Activity of D-ala-D-ala Ligase

The E. coli strain ST640 is a thermosensitive mutant exhibiting adeficient D-ala-D-ala ligase activity (Lugtenberg et al., 1973, J.Bacteriol. 113: 96-104). The plasmids pUC18 and pAT214 were introducedinto E. coli ST640 by transformation. The strains ST640 and ST640(pUC18) grew normally only at the permissive temperature (30° C.)whereas E. coli ST640 (pAT214) grew both at the permissive temperatureand at the non-permissive temperature (42° C.).

This test shows that VANA is functionally related to the D-Ala-D-Alaligases in E. coli and is probably capable of catalysing the sameligation reaction as DALIG.

II—VanS-VanR Two-Component Regulation System for the Control of theSynthesis of Depsipeptides of the Precursor of Peptidoglycans

Materials and Methods

Strains, Plasmids and Conditions of Culture

The restriction fragments of pIP816 (Tra⁻, Mob⁺, Vm^(r)) were cloned inderivatives of the vector pAT29 which constitutes a shuttle vectorbetween the Gram-positive and Gram-negative bacteria (oriR pAMβ1, oriRpUC, oriT RK2, spc, lacZ) (Trieu-Cuot et al., 1990, Nucleic Acids Res.18:4296). This vector was constructed by the inventors and used totransform the strain E. coli JM103 ((lac-proAB), supE, thi, strA,sbcB15, endA, hspR4, F traD36, proAB, LacI^(q), lacZ M15) (Messing etal., 1983, Methods Ezymol. 101:20-78). The plasmid DNA was prepared byan alkaline lysis protocol on a small scale (Sambrook et al., 1982,Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor N.Y.) and introduced by electroporation (Cruz-Rodz A.L. et al., 1990, Mol. Gen. Genet. 224: 152-154) in E. faecalis JH2-2(Fus^(R), Rif^(R)) (Jacob A. E. et al., 1974, J. Bacteriol. 117:360-372), by using a Gene Pulser apparatus (Bio-Rad Laboratories,Richmond, Calif.). The restriction profiles of the purified plasmidsfrom E. faecalis and E. coli were compared in order to detect possiblerearrangements of DNA.

The integrative plasmid pAT113 (Mob⁺, Em^(R), Km^(R), oriR PACYC184,attTn1545, LacZ) (Trieu-Cuot et al., Gene 106: 21-27) carries the joinedends of the transposon Tn1545. This vector does not replicate inGram-positive bacteria but is integrated into the chromosome of the hostby illegitimate recombination mediated by the integrase of Tn1545 or ofTn916 (Trieu-Cuot et al. previously mentioned). The integrative plasmidswere introduced into E. faecalis BM4148 (strain JH2-2::Tn916) by meansof electroporation. This strain is modified by the transposon Tn917described by Franque A. E. et al. (1981, J. Bacteriol. 145: 494-502).

The cultures were grown in brain-heart broth (BHI—Brain Heart InfusionBroth) or on agar at 37° C. The method of Steers et al (Antibiot.Chemother. Basel. 9: 307-311) was used to determine the minimalinhibitory concentrations (MICs) of the antibiotics on a Mueller-Hintongelose agar medium.

Recombinant DNA Procedures

The cleavage of DNA with restriction endonucleases (Boehringer Mannheimand Pharmacia), the purification of the DNA restriction fragments fromagarose gels, the conversion of the cohesive ends to blunt ends with theKlenow fragment of the DNA polymerase I of E. coli (BoehringerMannheim), the dephosphorylation of the ends of the DNA with calfintestinal phosphatase (Boehringer Mannheim), the ligation of the DNAfragments with the T4 DNA ligase (Amersham) were carried out accordingto the standard methods of Sambrook et al (1982, Molecular Cloning, aLaboratory Manual. Cold Spring Harbor Laboratory. Cold Spring HarborN.Y.).

Construction of Plasmids

The origin of the vectors and the inserts used for the recombinantplasmids constructed here is the following:

-   -   (i) vector pAT78 for the recognition of the promoter: the        amplified DNA of the cat gene for chloramphenicol        acetyltransferase of the plasmid pC194 of Staphylococcus aureus        (Horinouchi et al., 1982, J. Bacteriol. 150: 815-825) was        inserted between the PstI and SphI restriction sites of the        shuttle vector pAT29. Amplification by means of the polymerase        chain reaction was carried out by means of primers A1 and A2        which were synthesized by the methoxy phoshoramidite method        (Mabilat et 1990 Plasmid 23: 27-34). The sequence of the primer        A1 (SEQ ID NO:50) (5′ GCTGCAGATAAAAATTTAGGAGG) is composed of a        PstI recognition site (underlined) and 18 bases (positions 6        to 23) of pC194 which include the ribosomal binding site (RBS;        AGGAGG positions 18 to 23) of the cat gene. The sequence of the        primer A2 (SEQ ID NO:51) (5′ CGCATGCTATTATAAAA GCCAGTC) contains        the SphI cleavage site (underlined) and is complementary        (positions 8 to 24) to 17 bases at the 3′ end of the cat gene.        The triplet ATT at positions 9 to 11 correponds to the TAA stop        codon of cat. The DNA fragments amplified with the primers A1        and A2 hence consist of an open reading frame (orf) and a        ribosomal binding site for CAT (positions 1234 to 1912 according        to the numbering of Horinouchi et al. (1982, J. Bacteriol. 150:        815-825) flanked by the PstI and SphI sites. The position 1234        is located at the interior of the loop of the secondary        structure of the mRNA which blocks translation in the absence of        chloramphenicol. Thus, the amplified sequence does not contain        the cat promoter nor the sequence complementary to the RBS which        is essential for the regulation of translation Ambulos, N. P. et        al., 1984, Gene 28: 171-176).    -   (ii) expression vector pAT79: the ClaI-BssHII fragment of 243 bp        bearing the P2 promoter of the aphA-3 gene of the enterococcal        plasmid pJH1 (Caillaud et al., 1987, Mol. Gen. Genet. 207:        509-513) was inserted between the EcoRI and SacI restriction        sites of pAT78.    -   (iii) plasmid pAT80 and its derivatives: the BglII-XbaI fragment        of 5.5 kb of pIP816 was inserted between the BamHI and XbaI        sites of pAT78. The resulting plasmid, designated as pAT80 was        partially digested with HincII and ligated with the EcoRV        fragment containing a gene related to the apha-I gene of the        transposon Tn903 (Oka A. et al., 1981, J. Mol. Biol.        147:217-226. This fragment contains the aphA-I gene which codes        for the 3′aminoglycoside phosphotransferase of type I conferring        resistance to kanamycin. The insertion of aphAI was carried out        at three different sites in pAT80, generating the plasmids        pAT81, pAT83 and pAT85. The cassettes BamHI and EcoRI containing        aphA-I were inserted at the BamHI (to form the plasmid pAT84)        and EcoRI (to form the plasmid pAT82) sites of pAT80.    -   (iv) plasmids pAT86, pAT87, pAT88 and pAT89; the plasmid pAT86        was constructed by cloning the EcoRI-SacII fragment of 2.803 bp        of pAT80 coding for VanH and VanA at a SmaI site of pAT79, pAT87        was obtained by inserting the EcoRI-XbaI fragment of 3.4 kb of        pAT80 upstream from the cat gene of the detection vector of        promoter pAT78. The plasmid pAT88 resulted from the ligation of        pAT78 digested with EcoRI and BamHI to the EcoRI-BamHI fragment        of 1.731 bp of pAT80. The BglII-AccI fragment (positions 1        to 2356) of pAT80 was inserted into the polylinker of the        integrative vector pAT113, generating pAT89.

Sub-Cloning in M13 and Sequencing

The DNA restriction fragments were subcloned in a polylinker ofreplicative derivatives of the bacteriophage M13, these derivativesbeing called mp18 and mp19 (Norrander et al., 1983, Gene 26:101-106). E.coli JM103 was transfected with the recombinant phages and asingle-stranded DNA was prepared. The sequencing of the nucleotides wascarried out according to the conditions described by Sanger et al.(Proc. Natl. Acad. Sci. USA, 1977, 74: 5463-5467) by using the modifiedT7 DNA polymerase (Sequenase, United States, Biochemical CorporationCleveland Ohio) and [α-³⁵S] dATP (Amersham). The reaction products wereresolved on gradient gels of polyacrylamide in a 6% buffer.

Enzymatic Test

The JH2-2 derivatives of E. faecalis were grown to an optical densityOD₆₀₀ of 0.7 in a BHI broth supplemented with spectinomycin (300 μg/ml).The cells were treated with lysozyme, lysed by sonication and the celldebris were centrifuged for 45 minutes at 100,000 g according to thedescription given by Courvalin et al. (1978, Antimicrob. AgentsChemother. 13:716-725). The formation of 5-thio-2-nitrobenzoate wasmeasured at 37° C. in the presence and in the absence of chloramphenicoland the specific CAT activity was expressed in micromole per minute andper milligram of proteins (Shaw et al., 1975, Methods Enzymol.43:737-755).

Results

The vanH and vanA genes of pIP816 were cloned in a plasmid pAT79 underthe control of the heterologous promoter P2 (Caillaud et al., 1987, Mol.Gen. Genet. 207:509-513) and the plasmid pAT86 formed did not conferresistance to vancomycin on the strain E. faecalis JH2-2. These genesare thus not sufficient for the synthesis of peptoglycan in the absenceof the antibiotic. Different restriction fragments of pIP816 were clonedin the vector pAT78. The BglII-XbaI fragment of 5.5 kb of pAT80 is thesmallest fragment obtained which conferred resistance to vancomycin.

Nucleotide Sequence of the VanR and VanS Genes

The sequence of the insert in pAT80 was determined on both strands ofthe DNA from the BglII site to the ATG initiation codon for thetranslation of VanH. Two open reading frames (orf) were detected withinthe sequence of 2475 bp: the first open reading frame extends from thenucleotide 386 to the nucleotide 1123; at position 431 a sequencecharacteristic of the RBS sequences in Gram-positive bacteria is found,6 base pairs upstream from the ATG initiation codon for translation(TGAAAGGGTG) (SEQ ID NO:52); the other initiation codons for translationin this orf are not preceded by this type of sequence. The sequence of693 bp extending from the ATG codon at position 431 to the TAA codon atposition 1124 is capable of coding for a protein of 231 amino acids witha molecular mass of 26,612 Da which is designated as VanR.

In the case of the second open reading frame (from nucleotide 1089 tonucleotide 2255) the amino acid sequence deduced from the firstinitiation codon in phase (TTG at position 1104) would code for aprotein of 384 amino acids having a molecular mass of 43,847 Da anddesignated as VanS. The TTG codon at position 1116 and the ATG codon atposition 1164 are in-phase initiation codons for translation preceded bysequences with low complementarity with the 3′OH terminus of the 16Ssub-unit of the rRNA of B. subtilis (GGGGGGTTGG-N8-TTG (SEQ ID NO:53)and AGAACGAAAA-N6-ATG, (SEQ ID NO:54) respectively.

Between the last codon of vanS and the initiation codon ATG for thetranslation of vanH a sequence of 217 bp is to be observed whichcontains a repeated reverse sequence of 17 bp. This sequence does notfunction as a terminator of strong transcription.

The comparison of the sequences obtained with data bases has shown thatthe conserved amino acid residues identified by Stock et al. (1989,Microbiol. Rev. 53:450-490) in the kinase domain of 16 HPK (HistidineProtein Kinase) were detected in the C-terminal part of VanS. VanS (SEQID NO:14) possesses two groups of hydrophobic amino acids in theN-terminal region. The histidine residue 164 of VAnS is aligned with theresidue His216 of PhoR (SEQ ID NO:14) (Makino et al., 1986, J. Mol.Biol. 192: 549-556) and His 243 of EnvZ (SEQ ID NO:42) (Comeau et al.,1985, 164:578-584) which are presumed sites of autophosphorylation inthese proteins.

Similarly, the amino acids 1 to 122 of VanR (SEQ ID NO:12) exhibitsimilarities with the effector domains of response regulators RR. Theaspartic acid 53 of VanR might be a phosphorylation site because thisresidue is aligned with Asp 57 of Che Y (SEQ ID NO:46) which isphosphorylated by HPK associated with CheA and corresponds to aninvariant position in other proteins of the RR type (Stock et alpreviously mentioned). VanR might belong to the sub-class OmpR-PhoB ofRR which activates the initiation of transcription mediated by the RNApolymerase containing the 70S factor of E. coli (Stock et al. previouslymentioned).

Inactivation of the Van Genes by Insertion

Cassettes of resistance to kanamycin inserted in the group of van genesin the plasmid pAT80 have shown the following: the insertion in vanRsuppresses resistance to vancomycin and chloramphenicol; VanR is anactivator of transcription necessary for the expression of the genes forresistance to vancomycin. The inactivation of vanS leads to a two-foldreduction of the minimal inhibitory concentration (MIC) ofchloramphenicol and to a three-fold reduction of the specific CATactivity but the minimal inhibitory concentration of vancomycin remainsunchanged. Hence, VanS is necessary to produce a high level oftranscription of the genes for resistance to vancomycin although it isnot required for the expression of the phenotype of resistance tovancomycin.

Derivatives of pAT80 bearing insertions in vanH (pAT83), vanA (pAT84) orin the region 1.0 kb downstream from vanA (pAT85) have made it possibleto obtain resistance to chloramphenicol but not to vancomycin. Thisdissociated phenotype corresponds to the inactivation of genes codingfor enzymes which synthesize the depsipeptide precursors necessary forthe assembly of the bacterial cell walls in the presence of vancomycin.

Downstream from the vanA gene the presence of an inactivated orf hasbeen detected in pAT85 in the region of the sequence of 365 bp after theTGA codon of vanA and before the SacII site and this orf contains anin-phase ATG initiation codon preceded by a RBS-like sequence. Thissequence codes for a protein necessary for resistance to theglycopeptide, designated as VanX and which comprises maximally about 330amino acids.

Trans-Activation of the Transcription of the Van Genes

The integrative plasmid pAT89 coding for VanR and VanS was introducedinto the chromosome of E. faecalis BM4138. The plasmid pAT87 bearing thegenes vanH, vanA and vanX cloned upstream from the cat gene lacking thepromoter for pAT78 conferred resistance to vancomycin on this strain butnot to E. faecalis, JH2-2. The level of expression of the cat gene ofpAT87 in the strains BM4138::pAT89 and JH2-2 indicated that VanRactivates the transcription of the reporter gene localized at the 3′ endof the group of van genes. Similar levels of CAT synthesis were observedfor pAT88 which bears a transcription fusion between the 5′ parts ofvanA and the cat gene. These results show that in E. faecalisBM4138::pAT89 (pAT87) VanR and VanS encoded in the chromosome activatein a trans manner the transcription of vanA, vanH and vanX of pAT87making possible the production of resistance to vancomycin.

Moreover, it has been observed that the expression of the gene wasessentially constitutive when vanR and vanS were borne by a multicopyplasmid pAT80 and weakly inducible by vancomycin when the genes for theregulatory proteins were present on the chromosome of the host.

III—Characterization of the Sequence of the VanC Gene of Enterococcusgallinarum BM4174

Definition and Use of Universal Primers for the Amplification of GenesCoding for D-Ala-D-Ala Ligases and Related Proteins Implicated inResistance to Vancomycin

The protein VanA necessary for the expression of a high level ofresistance to the glycopeptides in E. faecium BM4147 shares a similarityof about 28 to 36% as regards its amino acids with the D-Ala-D-Alaligases of E. coli but possesses a different substrate specificity fromthat of these ligases. Peptides designated as 1 and 2 which areconserved in the sequences of the Dd1A and Dd1B ligases (Zawadzke, 1991Biochemistry 30:1673-1682) of E. coli and in the protein VanA wereselected in order to synthesize universal primers intended to amplifyinternal fragments of genes coding for D-Ala-D-Ala ligases or relatedenzymes. The peptide targets GEDG(S/T) (I/L)QG and NT(I/L)PGFT weretranslated back as is shown in Figure IV.1 in order to obtain degenerateoligonucleotides V1 and V2. As the peptides 1 and 2 of VanA, Dd1A andDd1B are separated by amino acid sequences of similar length, thepredicted size for the amplification product was about 640 bp.

Amplification by means of PCR with the DNA of E. coli JM83 and of E.faecium BM4147 made it possible to amplify products corresponding to theexpected size which have then been purified and cloned in thebacteriophage M13 mp10 (Norrander et al., 1983, Gene 26:101-106). Thesequencing of the insert obtained with E. coli JM83 has shown that theproduct of PCR was an internal fragment of dd1A. A probe generatedstarting from a recombinant phage obtained with the amplificationfragment of BM4147 was used for the Southern blot analysis of a DNA ofBM4147 and BM4147-1 which is a derivative of BM4147 sensitive tovancomycin and which lacks the plasmid pIP816 (Leclercq et al., 1988, N.Engl. J. Med. 319:157-161). The probe hybridized with the EcoRI DNAfragment of 4 kb from BM4147 but not with the DNA from E. faeciumBM4147-1. As the vanA gene is borne by the EcoRI fragment of 4 kb frompIP816, these results indicate that the primers also make possible theamplification of B part of vanA. Thus the oligonucleotides V1 and V2 mayamplify fragments of genes coding for different proteins related to theD-Ala-D-Ala ligases, and may do this in different species.

Amplification, Cloning and Sequencing of the VanC Gene

Amplification by means of PCR was carried out on the total DNA of E.gallinarum BM4174 and the amplification product obtained of about 640 bpwas cloned in the bacteriophage M13 mp10. The single-stranded DNAisolated from the recombinant phage was used to construct a probe C (Huet al., 1982, Gene 17:2171-2177). In Southern analysis the probehybridized with a PstI fragment of 1.7 kb from BM4174 but not with theDNA of BM4147 and BM4147-1.

The DNA of BM4174 was digested with PstI and fragments of 1.5 and 2 kbwere purified by electrophoresis on agarose gel and cloned in pUC18(Norrander et al., 1983, mentioned previously). The recombinant plasmidswere introduced into E. coli JM83 by transformation and screened byhybridization on colonies (Sambrook et al., 1989, Molecular cloning, alaboratory manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.) by using the probe C. A homology was detected with atransformant harbouring a plasmid called pAT216 which contained a PstIinsert of 1.7 kb. The sequence of the SacI-PstI part of 1347 bp of theinsert of pAT216 was determined on both strands of the DNA. The locationof the termination codons in the three reading frames of each strand ofDNA revealed the presence of an ORF phase located between the TGA codonsat positions 47 and 1244. The initiation codon of transcription ATG atposition 215 is preceded by a sequence GAAAGGAAGA characteristic of theRBS sequences complementary to the RNA of the 16S subunit of B. subtilis(Moran et al., 1982, Mol. Gen. Genet. 186:339-346). The sequence of 1029bp which extends from the ATG codon at position 215 to the TGA codon atposition 1244 might code for a protein of 343 amino acids having acalculated molecular mass of 37504 Da designated as VanC. A sequencehomology was detected between VanC, VanA and the D-Ala-D-Ala ligases ofE. coli. In particular, four domains of strong homology previously foundbetween VanA and the D-Ala-D-Ala ligases of the enterobacteria are alsopresent in VanC. The percentage of identical amino acids calculated forthese proteins taken two at a time varied between 29 and 38%. Thealignment of the four sequences revealed the presence of 57 invariantamino acids which include the conserved residues of the peptides 1 and 2used to define the oligonucleotide probes V1 and V2.

Inactivation of the VanC Gene by Insertion

In order to evaluate the contribution of vanC to resistance tovancomycin in E. gallinarum BM4174, the vanC gene was inactivated byinsertion. A EcoRI-HincII fragment of 690 bp, internal to vanC wascloned in pAT114 which does not replicate in Gram-positive bacteria. Theresulting pAT217 plasmid was introduced into BM4174 by electroporation(Cruz-Rodz et al., 1990, Mol. Gen. Genet. 224:152-154) and the clonessupposed to result from a homologous recombination leading to theintegration of pAT217 into vanC were selected on erythromycin. The cloneBM4175 was compared with BM4174 by Southern hybridization using theprobe C and aphA-3 specific for pAT114. The two probes hybridized withthe EcoRI fragment of 8.6 kb from BM4175. The probe C hybridized with afragment of 2.5 kb from BM4174 whereas no signal was observed with theprobe aphA-3. The results indicate that the plasmid pAT217 of 6.1 kb wasintegrated into the vanC gene. The determination of the minimalinhibitory concentration of vancomycin for BM4174 (16 mg/l) and BM4175(2 mg/l) indicated that the inactivation by insertion in vanC abolishesresistance to vancomycin.

VanC is thus required for resistance to vancomycin. It may thus besupposed that this protein synthesizes a dipeptide or a depsipeptidewhich is incorporated into the precursors of peptidoglycans and is notrecognized by vancomycin.

The sequences which are the object of the invention are given in thefollowing pages after the list of the sequences containing thedescription of these sequences. In this list of the sequences, theproteins are identified with respect to the position of the nucleotidebases corresponding to the amino acids of the extremities of theproteins.

LIST OF THE SEQUENCES

(contained in the sequences I (Ia, Ib), II presented below or in thesequence shown in FIG. 5).

Amino Acid Sequences

SEQ ID NO 2 (VanH): sequence of the first resistance protein,corresponding to the amino acid sequence of the open reading frame No.3, starting at the base 3501 and terminating at the base 4529,containing the sequence coding for the vanH gene between the bases 3564and 4529 with respect to the sequence shown in FIG. 5 or correspondingto the sequence between the positions of the nucleotides 6018 and 6983of the sequence Ia.

SEQ ID NO 4 (VanA): sequence of the VanA protein, corresponding to theamino acid sequence of the open reading frame No. 1, starting at thebase 4429 and terminating at the base 5553 with respect to the sequenceshown in FIG. 5 or corresponding to the sequence between the positionsof the nucleotides 6977 and 7807 of the sequence Ia.

SEQ ID NO 6 (VanX): sequence of the third resistance protein,corresponding to the amino acid sequence of the open reading frame No.3, starting at the base 5526 and terminating at the base 6167 withrespect to the sequence shown in FIG. 5 or corresponding to the sequencebetween the positions of the nucleotides 7816 and 8621 of the sequenceIa.

SEQ ID NO 12 (VanR) sequence of the regulatory protein R, correspondingto the amino acid sequence of the open reading frame No. 1, starting atthe base 1477 and terminating at the base 2214 with respect to thesequence shown in FIG. 5 or corresponding to the sequence between thepositions of the nucleotides 3976 and 4668 of the sequence Ia.

SEQ ID NO 14 (VanS): sequence of the sensor protein S, corresponding tothe amino acid sequence of the open reading frame No. 2, starting at thebase 2180 and terminating at the base 3346 with respect to the sequenceshown in FIG. 5 or corresponding to the sequence between the positionsof the nucleotides 4648 and 5800 of the sequence Ia.

SEQ ID NO 19: sequence of the transposase corresponding to the aminoacids included between the nucleotides 150 and 3112 of the sequence Ib.

SEQ ID NO 21: sequence of the resolvase comprising the amino acidssituated between the positions of the nucleotides 3187 and 3759 of thesequence Ia.

SEQ ID NO 23: VanY sequence comprising the amino acids situated betweenthe positions of the nucleotides 9046 and 9960 of the sequence Ia.

SEQ ID NO 25: VanZ sequence comprising the amino acids situated betweenthe positions of the nucleotides 10116 and 10598 of the sequence Ia.

SEQ ID NO 8: VanC amino acid sequence shown in list II.

Nucleotide Sequences

SEQ ID NO 15: nucleotide sequence containing the sequence coding for the5 proteins as well as the flanking sequences, shown in FIG. 5.

SEQ ID NO 17: sequence containing the sequence coding for the 3resistance proteins as well as the flanking sequences and starting atthe base 3501 and terminating at the base 6167, shown in FIG. 5.

SEQ ID NO 1: sequence of the vanA gene, starting at the base 4429 andterminating at the base 5553 of the sequence shown in FIG. 5, orcorresponding to the nucleotide sequence situated between thenucleotides 6977 and 7807 of the sequence Ia.

SEQ ID NO 1: sequence coding for the first resistance protein calledVanH, starting at the base 3501 and terminating at the base 4529, inparticular the sequence vanH, the coding sequence of which is locatedbetween the bases 3564 and 4529 of the sequence shown in FIG. 5, orcorresponding to the nucleotide sequence situated between thenucleotides 6018 and 6983 of the sequence Ia.

SEQ ID NO 5: sequence coding for the third resistance protein VanX,starting at the base 5526 and terminating at the base 6167 of thesequence shown in FIG. 5, or corresponding to the nucleotide sequencesituated between the nucleotides 7816 and 8621 of the sequence Ia.

SEQ ID NO 16: sequence of the transposon coding for the transposase, theresolvase, vanR, VAnS, VanH, VanA, VanX, VanY and VanZ and containingthe repeated reverse sequence of 38 bp at its N- and C-termini andcorresponding to the sequence Ia.

SEQ ID NO 18: sequence coding for the transposase, starting at the base150 and terminating at the base 3112 of the sequence Ib.

SEQ ID NO 20: sequence coding for the resolvase, starting at the base3187 and terminating at the base 3759 of the sequence Ia.

SEQ ID NO 22: sequence coding for VanY, starting at the base 9046 andterminating at the base 9960 of the sequence Ia.

SEQ ID NO 24: sequence coding for VanZ, starting at the base 10116 andterminating at the base 10598 of the sequence Ia.

SEQ ID NO 7: sequence coding for VanC, shown in the list II in relationto the protein VanC.

SEQ ID NO 16: complete sequence Ia of the transposon of E. faecium,starting at the base 1 and terminating at the base 10851.

SEQ ID NO 11: sequence coding for the protein VanR, starting at the base3976 and terminating at the base 4668 of the sequence Ia.

SEQ ID NO 13: sequence coding for the protein VanS, starting at the base4648 and terminating at the base 5800 of the sequence Ia.

I. Nucleotide sequence of the transposon and translation Ia. (+) Strand1 GGG GTA GCG TCA GGA AAA TGC GGA TTT ACA ACG CTA AGC CTA TTT TCC TGACGA ATC CCT 61 CGT TTT TAA CAA CGT TAA GAA AGT TTT AGT GGT CTT AAA GAATTT AAT GAG ACT ACT TTC 121 TCT GAG TTA AAA TGG TAT TCT CCT AGT AAA TTAATA TGT TCC CAA CCT AAG GGC GAC ATA 181 TGG TGT AAC AAA TCT TCA TTA AAGCTA CCT GTC CGT TTT TTA TAT TCA ACT GCT GTT GTT 241 AGG TGG AGA GTA TTCCAA ATA CTT ATA GCA TTG ATA ATT ATG TTT AAA GCA CTG GCT CTT 301 TGC AATTGA TGC TGT ATG GTG CGT TCT CTA AGC TCA CCT TGT TTT CCG AAG AAA ATA GCT361 CTT GCC AAT CCA TTC ATG GCT TCT CCT TTA TTC AAT CCT CTT TGT ATT TTTCTT CTT AAT 421 GAT TCA TCC GAT ATA TAA TTC AAA ATA AAG ATC GTT TTT TCTATT CGG CCC ATC TCA CGT 481 AAG GCT GTA GCT AAG CTG TTT TGT CTT GAA TAGGAA CCT AGC TTC CCC ATA ATA AGG GAT 541 GCT GAA ACT GTT CCC TCC CTT ATAGAA TGA GCT AAT CGC AAA ACA TCC TCA TAA TTT TCT 601 TTA ATG ACC TTT GTATTT ATT TGT CCA CGT AAA ATG GCT TCT AGT TTT GGA TAC TCA CTT 661 GCT TTATCT ATC GTA AAT AAT TTT GAG TCC GAT AAA TCC CTT ATT CTT GGG GCA AAT TTA721 AAT CCT AAT AAA TGA GTC AGT CCG AAT ATT TGG TCA GTG TAA CCG GCA GTGTCT GTA TAA 781 TGT TCC TCT ATG TTT AGA TCC GTC TCA TGA TGT AAC AAA CCATCC AAA ACA TGA ATC GCA 841 TCT CTT GAA TTA GTA TGA ATA ATC TTT GTG TAGTAA GAA GAG AAT TGA TCA CTT GTA AAT 901 CGG TAG ATG GTG GCT CCT TTT CCAGTT CCA TAA TGT GGA TTT GCA TCT GCA TGT AGT GAT 961 GAA ACA CCT AGC TGCATT CTC ATA CCA TCT GAC GAA GAT GTT GTA CCG TCG CCC CAA TAG 1021 AAA GGCAAT TGT AAT TTA TGA TGA AAG TTT ACT AAT ATG GCT TGG GCT TTA TTC ATG GCA1081 TCT TCA TAC ATG CGC CAT TGA GAT ACA TTG GCT AGT TGC TTA TAT GTA AGTCCG GGT GTG 1141 GCT TCG GCC ATC TTG CTC AAG CCA ATA TTC ATT CCC ATT CCTAAA AGG GCA GCC ATG ATA 1201 ATG ATT GTT TCT TCC TTA TCT GGT TTT CGA TTATTG GAA GCA TGA GTG AAT TGC TCA TGA 1261 AAT CCT GTT ATA TGG GCC ACA TCCATG AGT AAA TCA GTT AAT TTT ATT CTT GGT AGC ATC 1321 TGA TAA AGG CTT GCACTA AAT TTT TTT GCT TCT TCT GGA ACA TCT TTT TCT AAG CGT GCA 1381 AGT GATAGC TTT CCT TTT TCA AGA GAA ACC CCA TCT AAC TTA TTG GAA TTG GCA GCT AAC1441 CAC TTT AAC CTT TCA TTA AAG CTG CTG GTT CTC TCC GTT ATA TAA TCT TCGAAT GAT AAA 1501 CTA ACT GAT AAT CTC GTA TTC CCC TTC GAT TGA TTC CAT GTATCT TCC GAA AAC AAA TAT 1561 TCC TCA AAA TCC CTA TAT TGT CTG CTG CCA ACAATG GAA ACA TCT CCT GCC CGA ACA TGC 1621 TCC CGA AGT TCT GTT AAA ACA GCCATT TCA TAG TAA TGA CGA TTA ATT GTT GTA CCA TCA 1681 TCC TCG TAT AAA TGTCTT TTC CAT CGT TTT GAA ATA AAA TCC ACA GGT GAG TCA TCA GGC 1741 ACT TTTCGC TTT CCA GAT TCG TTC ATT CCT CGG ATA ATC TCA ACA GCT TGT AAA AGT GGC1801 TCA TTT GCC TTT GTA GAA TGA AAT TCC AAT ACT CTT AAT AGC GTT GGC GTATAT TTT CTT 1861 AGT GAA TAA AAC CGT TTT TGC AGT AAG TCT AAA TAA TCA TAGTCG GCA GGA CGT GCA AGT 1921 TCC TGA GCC TCT TCT ACT GAA GAG ACA AAG GTATTC CAT TCA ATA ACC GAT TCT AAA ACC 1981 TTA AAA ACG TCT AAT TTT TCC TCTCTT GCT TTA ATT AAT GCT TGT CCG ATG TTC GTA AAG 2041 TGT ATA ACT TTC TCATTT AGC TTT TTA CCG TTT TGT TTC TGG ATT TCC TCT TGA GCC TTA 2101 CGA CCTTTT GAT AAC AAA CTA AGT ATT TGC CTA TCA TGA ATT TCA AAC GCT TTA TCC GTT2161 AGC TCC TGA GTA AGT TGT AAT AAA TAG ATG GTT AAT ATC GAA TAA CGT TTATTT TCT TGA 2221 AAG TCA CGG AAT GCA TAC GGC TCG TAT CTT GAG CCT AAG CGAGAC AGC TGC AAC AGG CGG 2281 TTA CGG TGC AAA TGA CTA ATT TGC ACT GTT TCTAAA TCC ATT CCT CGT ATG TAT TCG AGT 2341 CGT TCT ATT ATT TTT AGA AAA GTTTCG GGT GAA GGA TGA CCC GGT GGC TCT TTT AAC CAA 2401 CCC AAT ATC GTT TTATTG GAT TCG GAT GGA TGC TGC GAG GTA ATA ATC CCT TCA AGC TTT 2461 TCT TTTTGC TCA TTT GTT AGA GAT TTA CTA ACC GTA TTA AAT AGC TTC TTT TCA GCC ATT2521 GCC CTT GCT TCC CAC ACC ATT CTT TCA AGT GTA GTG ATA GCA GGC AGT ATAATT TTG TTT 2581 TTT CTT AGA AAA TCT ATG CAT TCA TGC AGT AGA TGA ATG GCATCA CCA TTT TCC AAA GCT 2641 AAT TGA TGA AGG TAC TTA AAT GTC ATT CGA TATTCA CTC AGG GTA AAA GTT ACA AAG TCG 2701 TAT TCA CTT CGA ATT TCT TTC AAATGA TCC CAA AGT GTA TTT TCC CTT TGA GGA TAA TGA 2761 TCA AGC GAG GAT GGACTA ACA CCA ATC TGT TTC GAT ATA TAT TGT ATG ACC GAA TCT GGG 2821 ATG CTTTTG ATA TGA GTG TAT GGC CAA CCG GGA TAC CGA AGA ACA GCT AAT TGA ACA GCA2881 AAT CCT AAA CGG TTT TCT TCC CTC CTT CGC TTA TTA ACT ATT TCT AAA TCCCGT TTG GAA 2941 AAA GTG AAG TAG GTC CCC AGT ATC CAT TCA TCT TCA GGG ATTTGC ATA AAA GCC TGT CTC 3001 TGT TCC GGT GTA AGC AAT TCT CTA CCT CTC GCAATT TTC ATT CAG TAT CAT TCC ATT TCT 3061 GTA TTT TCA ATT TAT TAG TTC AATTAT ATA TCA ATA GAG TGT ACT CTA TTG ATA CAA ATG 3121 TAG TAG ACT GAT AAAATC ATA GTT AAG AGC GTC TCA TAA GAC TTG TCT CAA AAA TGA GGT 3181         resolvase         LEU ARG LYS ILE GLY TYR ILE ARG VAL SER SERTHR ASN GLN ASN PRO SER ARG GAT ATT TTG CGG AAA ATC GGT TAT ATT CGT GTCAGT TCG ACT AAC CAG AAT CCT TCA AGA 3241 GLN PHE GLN GLN LEU ASN GLU ILEGLY MET ASP ILE ILE TYR GLU GLU LYS VAL SER GLY CAA TTT CAG CAG TTG AACGAG ATC GGA ATG GAT ATT ATA TAT GAA GAG AAA GTT TCA GGA 3301 ALA THR LYSASP ARG GLU GLN LEU GLN LYS VAL LEU ASP ASP LEU GLN GLU ASP ASP ILE GCAACA AAG GAT CGC GAG CAA CTT CAA AAA GTG TTA GAC GAT TTA CAG GAA GAT GACATC 3361 ILE TYR VAL THR ASP LEU THR ARG ILE THR ARG SER THR GLN ASP LEUPHE GLU LEU ILE ATT TAT GTT ACA GAC TTA ACT CGA ATC ACT CGT AGT ACA CAAGAT CTA TTT GAA TTA ATC 3421 ASP ASN ILE ARG ASP LYS LYS ALA SER LEU LYSSER LEU LYS ASP THR TRP LEU ASP LEU GAT AAC ATA CGA GAT AAA AAG GCA AGTTTA AAA TCA CTA AAA GAT ACA TGG CTT GAT TTA 3481 SER GLU ASP ASN PRO TYRSER GLN PHE LEU ILE THR VAL MET ALA GLY VAL ASN GLN LEU TCA GAA GAT AATCCA TAC AGC CAA TTC TTA ATT ACT GTA ATG GCT GGT GTT AAC CAA TTA 3541 GLUARG ASP LEU ILE ARG MET ARG GLN ARG GLU GLY ILE GLU LEU ALA LYS LYS GLUGLY GAG CGA GAT CTT ATT CGG ATG AGA CAA CGT GAA GGG ATT GAA TTG GCT AAGAAA GAA GGA 3601 LYS PHE LYS GLY ARG LEU LYS LYS TYR HIS LYS ASN HIS ALAGLY MET ASN TYR ALA VAL AAG TTT AAA GGT CGA TTA AAG AAG TAT CAT AAA AATCAC GCA GGA ATG AAT TAT GCG GTA 3661 LYS LEU TYR LYS GLU GLY ASN MET THRVAL ASN GLN ILE CYS GLU ILE THR ASN VAL SER AAG CTA TAT AAA GAA GGA AATATG ACT GTA AAT CAA ATT TGT GAA ATT ACT AAT GTA TCT 3721 ARG ALA SER LEUTYR ARG LYS LEU SER GLU VAL ASN ASN AGG GCT TCA TTA TAC AGG AAA TTA TCAGAA GTG AAT AAT TAG CCA TTC TGT ATT CCG CTA 3781 ATG GGC AAT ATT TTT AAAGAA GAA AAG GAA ACT ATA AAA TAT TAA CAG CCT CCT AGC GAT 3841 GCC GAA AAGCCC TTT GAT AAA AAA AGA ATC ATC ATC TTA AGA AAT TCT TAG TCA TTT ATT 3901ATG TAA ATG CTT ATA AAT TCG GCC CTA TAA TCT GAT AAA TTA TTA AGG GCA AACTTA TGT 3961              VanR  MET SER ASP LYS ILE LEU ILE VAL ASP ASPGLU HIS  GLU ILE ALA GAA AGG GTG ATA ACT ATG AGC GAT AAA ATA CTT ATT GTGGAT GAT GAA CAT GAA ATT GCC 4021 ASP LEU VAL GLU LEU TYR LEU LYS ASN GLUASN TYR THR VAL PHE LYS TYR TYR THR ALA GAT TTG GTT GAA TTA TAC TTA AAAAAC GAG AAT TAT ACG GTT TTC AAA TAC TAT ACC GCC 4081 LYS GLU ALA LEU GLUCYS ILE ASP LYS SER GLU ILE ASP LEU ALA ILE LEU ASP ILE MET AAA GAA GCATTG GAA TGT ATA GAC AAG TCT GAG ATT GAC CTT GCC ATA TTG GAC ATC ATG 4141LEU PRO GLY THR SER GLY LEU THR ILE CYS GLN LYS ILE ARG ASP LYS HIS THRTYR PRO CTT CCC GGC ACA AGC GGC CTT ACT ATC TGT CAA AAA ATA AGG GAC AAGCAC ACC TAT CCG 4201 ILE ILE MET LEU THR GLY LYS ASP THR GLU VAL ASP LYSILE THR GLY LEU THR ILE GLY ATT ATC ATG CTG ACC GGG AAA GAT ACA GAG GTAGAT AAA ATT ACA GGG TTA ACA ATC GGC 4261 ALA ASP ASP TYR ILE THR LYS PROPHE ARG PRO LEU GLU LEU ILE ALA ARG VAL LYS ALA GCG GAT GAT TAT ATA ACGAAG CCC TTT CGC CCA CTG GAG TTA ATT GCT CGG GTA AAG GCC 4321 GLN LEU ARGARG TYR LYS LYS PHE SER GLY VAL LYS GLU GLN ASN GLU ASN VAL ILE VAL CAGTTG CGC CGA TAC AAA AAA TTC AGT GGA GTA AAG GAG CAG AAC GAA AAT GTT ATCGTC 4381 HIS SER GLY LEU VAL ILE ASN VAL ASN THR HIS GLU CYS TYR LEU ASNGLU LYS GLN LEU CAC TCC GGC CTT GTC ATT AAT GTT AAC ACC CAT GAG TGT TATCTG AAC GAG AAG CAG TTA 4441 SER LEU THR PRO THR GLU PHE SER ILE LEU ARGILE LEU CYS GLU ASN LYS GLY ASN VAL TCC CTT ACT CCC ACC GAG TTT TCA ATACTG CGA ATC CTC TGT GAA AAC AAG GGG AAT GTG 4501 VAL SER SER GLU LEU LEUPHE HIS GLU ILE TRP GLY ASP GLU TYR PHE SER LYS SER ASN GTT AGC TCC GAGCTG CTA TTT CAT GAG ATA TGG GGC GAC GAA TAT TTC AGC AAG AGC AAC 4561 ASNTHR ILE THR VAL HIS ILE ARG HIS LEU ARG GLU LYS MET ASN ASP THR ILE ASPASN AAC ACC ATC ACC GTG CAT ATC CGG CAT TTG CGC GAA AAA ATG AAC GAC ACCATT GAT AAT 4621 PRO LYS TYR ILE LYS THR VAL TRP GLYVALGLYTYRLYSILEGLULYS CCG AAA TAT ATA AAA ACG GTA TGG GGGGTTGGTTATAAAATTGAAAAAT AAA AAA AAC GAC                            VanS    LEUVALILELYSLEULYSASN  LYS LYS ASNASP 4682 TYR SER LYS LEU GLU ARG LYS LEU TYR MET TYR ILE VAL ALA ILE VALVAL VAL ALA ILE TAT TCC AAA CTA GAA CGA AAA CTT TAC ATG TAT ATC GTT GCAATT GTT GTG GTA GCA ATT 4742 VAL PHE VAL LEU TYR ILE ARG SER MET ILE ARGGLY LYS LEU GLY ASP TRP ILE LEU SER GTA TTC GTG TTG TAT ATT CGT TCA ATGATC CGA GGG AAA CTT GGG GAT TGG ATC TTA AGT 4802 ILE LEU GLU ASN LYS TYRASP LEU ASN HIS LEU ASP ALA MET LYS LEU TYR GLN TYR SER ATT TTG GAA AACAAA TAT GAC TTA AAT CAC CTG GAC GCG ATG AAA TTA TAT CAA TAT TCC 4862 ILEARG ASN ASN ILE ASP ILE PHE ILE TYR VAL ALA ILE VAL ILE SER ILE LEU ILELEU ATA CGG AAC AAT ATA GAT ATC TTT ATT TAT GTG GCG ATT GTC ATT AGT ATTCTT ATT CTA 4922 CYS ARG VAL MET LEU SER LYS PHE ALA LYS TYR PHE ASP GLUILE ASN THR GLY ILE ASP TGT CGC GTC ATG CTT TCA AAA TTC GCA AAA TAC TTTGAC GAG ATA AAT ACC GGC ATT GAT 4982 VAL LEU ILE GLN ASN GLU ASP LYS GLNILE GLU LEU SER ALA GLU MET ASP VAL MET GLU GTA CTT ATT CAG AAC GAA GATAAA CAA ATT GAG CTT TCT GCG GAA ATG GAT GTT ATG GAA 5042 GLN LYS LEU ASNTHR LEU LYS ARG THR LEU GLU LYS ARG GLU GLN ASP ALA LYS LEU ALA CAA AAGCTC AAC ACA TTA AAA CGG ACT CTG GAA AAG CGA GAG CAG GAT GCA AAG CTG GCC5102 GLU GLN ARG LYS ASN ASP VAL VAL MET TYR LEU ALA HIS ASP ILE LYS THRPRO LEU THR GAA CAA AGA AAA AAT GAC GTT GTT ATG TAC TTG GCG CAC GAT ATTAAA ACG CCC CTT ACA 5162 SER ILE ILE GLY TYR LEU SER LEU LEU ASP GLU ALAPRO ASP MET PRO VAL ASP GLN LYS TCC ATT ATC GGT TAT TTG AGC CTG CTT GACGAG GCT CCA GAC ATG CCG GTA GAT CAA AAG 5222 ALA LYS TYR VAL HIS ILE THRLEU ASP LYS ALA TYR ARG LEU GLU GLN LEU ILE ASP GLU GCA AAG TAT GTG CATATC ACG TTG GAC AAA GCG TAT CGA CTC GAA CAG CTA ATC GAC GAG 5282 PHE PHEGLU ILE THR ARG TYR ASN LEU GLN THR ILE THR LEU THR LYS THR HIS ILE ASPTTT TTT GAG ATT ACA CGG TAT AAC CTA CAA ACG ATA ACG CTA ACA AAA ACG CACATA GAC 5342 LEU TYR TYR MET LEU VAL GLN MET THR ASP GLU PHE TYR PRO GLNLEU SER ALA HIS GLY CTA TAC TAT ATG CTG GTG CAG ATG ACC GAT GAA TTT TATCCT CAG CTT TCC GCA CAT GGA 5402 LYS GLN ALA VAL ILE HIS ALA PRO GLU ASPLEU THR VAL SER GLY ASP PRO ASP LYS LEU AAA CAG GCG GTT ATT CAC GCC CCCGAG GAT CTG ACC GTG TCC GGC GAC CCT GAT AAA CTC 5462 ALA ARG VAL PHE ASNASN ILE LEU LYS ASN ALA ALA ALA TYR SER GLU ASP ASN SER ILE GCG AGA GTCTTT AAC AAC ATT TTG AAA AAC GCC GCT GCA TAC AGT GAG GAT AAC AGC ATC 5522ILE ASP ILE THR ALA GLY LEU SER GLY ASP VAL VAL SER ILE GLU PHE LYS ASNTHR GLY ATT GAC ATT ACC GCG GGC CTC TCC GGG GAT GTG GTG TCA ATC GAA TTCAAG AAC ACT GGA 5582 SER ILE PRO LYS ASP LYS LEU ALA ALA ILE PHE GLU LYSPHE TYR ARG LEU ASP ASN ALA AGC ATC CCA AAA GAT AAG CTA GCT GCC ATA TTTGAA AAG TTC TAT AGG CTG GAC AAT GCT 5642 ARG SER SER ASP THR GLY GLY ALAGLY LEU GLY LEU ALA ILE ALA LYS GLU ILE ILE VAL CGT TCT TCC GAT ACG GGTGGC GCG GGA CTT GGA TTG GCG ATT GCA AAA GAA ATT ATT GTT 5702 GLN HIS GLYGLY GLN ILE TYR ALA GLU SER ASN ASP ASN TYR THR THR PHE ARG VAL GLU CAGCAT GGA GGG CAG ATT TAC GCG GAA AGC AAT GAT AAC TAT ACG ACG TTT AGG GTAGAG 5762 LEU PRO ALA MET PRO ASP LEU VAL ASP LYS ARG ARG SER CTT CCA GCGATG CCA GAC TTG GTT GAT AAA AGG AGG TCC TAA GA GAT GTA TAT AAT TTT 5821TTA GGA AAA TCT CAA GGT TAT CTT TAC TTT TTC TTA GGA AAT TAA CAA TTT AATATT AAG 5881 AAA CGG CTC GTT CTT ACA CGG TAG ACT TAA TAC CGT AAG AAC GAGCCG TTT TCG TTC TTC 5941 AGA GAA AGA TTT GAC AAG ATT ACC ATT GGC ATC CCCGTT TTA TTT GGT GCC TTT CAC AGA 6001        VanH         MET ASN ASN ILEGLY ILE THR VAL TYR GLY CYS  GLU GLN ASP GLU AAGGGTTGG TCT TAA TT ATGAAT AAC ATC GGC ATT ACT GTT TAT GGA TGT GAG CAG GAT GAG 6063 ALA ASP ALAPHE HIS ALA LEU SER PRO ARG PHE GLY VAL MET ALA THR ILE ILE ASN ALA GCAGAT GCA TTC CAT GCT CTT TCG CCT CGC TTT GGC GTT ATG GCA ACG ATA ATT AACGCC 6123 ASN VAL SER GLU SER ASN ALA LYS SER ALA PRO PHE ASN GLN CYS ILESER VAL GLY HIS AAC GTG TCG GAA TCC AAC GCC AAA TCC GCG CCT TTC AAT CAATGT ATC AGT GTG GGA CAT 6183 LYS SER GLU ILE SER ALA SER ILE LEU LEU ALALEU LYS ARG ALA GLY VAL LYS TYR ILE AAA TCA GAG ATT TCC GCC TCT ATT CTTCTT GCG CTG AAG AGA GCC GGT GTG AAA TAT ATT 6243 SER THR ARG SER ILE GLYCYS ASN HIS ILE ASP THR THR ALA ALA LYS ARG MET GLY ILE TCT ACC CGA AGCATC GGC TGC AAT CAT ATA GAT ACA ACT GCT GCT AAG AGA ATG GGC ATC 6303 THRVAL ASP ASN VAL ALA TYR SER PRO ASP SER VAL ALA ASP TYR THR MET MET LEUILE ACT GTC GAC AAT GTG GCG TAC TCG CCG GAT AGC GTT GCC GAT TAT ACT ATGATG CTA ATT 6363 LEU MET ALA VAL ARG ASN VAL LYS SER ILE VAL ARG SER VALGLU LYS HIS ASP PHE ARG CTT ATG GCA GTA CGC AAC GTA AAA TCG ATT GTG CGCTCT GTG GAA AAA CAT GAT TTC AGG 6423 LEU ASP SER ASP ARG GLY LYS VAL LEUSER ASP MET THR VAL GLY VAL VAL GLY THR GLY TTG GAC AGC GAC CGT GGC AAGGTA CTC AGC GAC ATG ACA GTT GGT GTG GTG GGA ACG GGC 6483 GLN ILE GLY LYSALA VAL ILE GLU ARG LEU ARG GLY PHE GLY CYS LYS VAL LEU ALA TYR CAG ATAGGC AAA GCG GTT ATT GAG CGG CTG CGA GGA TTT GGA TGT AAA GTG TTG GCT TAT6543 SER ARG SER ARG SER ILE GLU VAL ASN TYR VAL PRO PHE ASP GLU LEU LEUGLN ASN SER AGT CGC AGC CGA AGT ATA GAG GTA AAC TAT GTA CCG TTT GAT GAGTTG CTG CAA AAT AGC 6603 ASP ILE VAL THR LEU HIS VAL PRO LEU ASN THR ASPTHR HIS TYR ILE ILE SER HIS GLU GAT ATC GTT ACG CTT CAT GTG CCG CTC AATACG GAT ACG CAC TAT ATT ATC AGC CAC GAA 6663 GLN ILE GLN ARG MET LYS GLNGLY ALA PHE LEU ILE ASN THR GLY ARG GLY PRO LEU VAL CAA ATA CAG AGA ATGAAG CAA GGA GCA TTT CTT ATC AAT ACT GGG CGC GGT CCA CTT GTA 6723 ASP THRTYR GLU LEU VAL LYS ALA LEU GLU ASN GLY LYS LEU GLY GLY ALA ALA LEU ASPGAT ACC TAT GAG TTG GTT AAA GCA TTA GAA AAC GGG AAA CTG GGC GGT GCC GCATTG GAT 6783 VAL LEU GLU GLY GLU GLU GLU PHE PHE TYR SER ASP CYS THR GLNLYS PRO ILE ASP ASN GTA TTG GAA GGA GAG GAA GAG TTT TTC TAC TCT GAT TGCACC CAA AAA CCA ATT GAT AAT 6843 GLN PHE LEU LEU LYS LEU GLN ARG MET PROASN VAL ILE ILE THR PRO HIS THR ALA TYR CAA TTT TTA CTT AAA CTT CAA AGAATG CCT AAC GTG ATA ATC ACA CCG CAT ACG GCC TAT 6903 TYR THR GLU GLN ALALEU ARG ASP THR VAL GLU LYS THR ILE LYS ASN CYS LEU ASP PHE TAT ACC GAGCAA GCG TTG CGT GAT ACC GTT GAA AAA ACC ATT AAA AAC TGT TTG GAT TTT 6963           VanA     METASN ARG ILE LYS VAL ALA ILE LEU PHE GLY  GLY CYSSER GAA AGG AGA CAG GAG CATGAAT AGA ATA AAA GTT GCA ATA CTG TTT GGG GGTTGC TCA GLU ARG ARG GLN GLU HISGLU 7021 GLU GLU HIS ASP VAL SER VAL LYSSER ALA ILE GLU ILE ALA ALA ASN ILE ASN LYS GLU GAG GAG CAT GAC GTA TCGGTA AAA TCT GCA ATA GAG ATA GCC GCT AAC ATT AAT AAA GAA 7081 LYS TYR GLUPRO LEU TYR ILE GLY ILE THR LYS SER GLY VAL TRP LYS MET CYS GLU LYS AAATAC GAG CCG TTA TAC ATT GGA ATT ACG AAA TCT GGT GTA TGG AAA ATG TGC GAAAAA 7141 PRO CYS ALA GLU TRP GLU ASN ASP ASN CYS TYR SER ALA VAL LEU SERPRO ASP LYS LYS CCT TGC GCG GAA TGG GAA AAC GAC AAT TGC TAT TCA GCT GTACTC TCG CCG GAT AAA AAA 7201 MET HIS GLY LEU LEU VAL LYS LYS ASN HIS GLUTYR GLU ILE ASN HIS VAL ASP VAL ALA ATG CAC GGA TTA CTT GTT AAA AAG AACCAT GAA TAT GAA ATC AAC CAT GTT GAT GTA GCA 7261 PHE SER ALA LEU HIS GLYLYS SER GLY GLU ASP GLY SER ILE GLN GLY LEU PHE GLU LEU TTT TCA GCT TTGCAT GGC AAG TCA GGT GAA GAT GGA TCC ATA CAA GGT CTG TTT GAA TTG 7321 SERGLY ILE PRO PHE VAL GLY CYS ASP ILE GLN SER SER ALA ILE CYS MET ASP LYSSER TCC GGT ATC CCT TTT GTA GGC TGC GAT ATT CAA AGC TCA GCA ATT TGT ATGGAC AAA TCG 7381 LEU THR TYR ILE VAL ALA LYS ASN ALA GLY ILE ALA THR PROALA PHE TRP VAL ILE ASN TTG ACA TAC ATC GTT GCG AAA AAT GCT GGG ATA GCTACT CCC GCC TTT TGG GTT ATT AAT 7441 LYS ASP ASP ARG PRO VAL ALA ALA THRPHE THR TYR PRO VAL PHE VAL LYS PRO ALA ARG AAA GAT GAT AGG CCG GTG GCAGCT ACG TTT ACC TAT CCT GTT TTT GTT AAG CCG GCG CGT 7501 SER GLY SER SERPHE GLY VAL LYS LYS VAL ASN SER ALA ASP GLU LEU ASP TYR ALA ILE TCA GGCTCA TCC TTC GGT GTG AAA AAA GTC AAT AGC GCG GAC GAA TTG GAC TAC GCA ATT7561 GLU SER ALA ARG GLN TYR ASP SER LYS ILE LEU ILE GLU GLN ALA VAL SERGLY CYS GLU GAA TCG GCA AGA CAA TAT GAC AGC AAA ATC TTA ATT GAG CAG GCTGTT TCG GGC TGT GAG 7621 VAL GLY CYS ALA VAL LEU GLY ASN SER ALA ALA LEUVAL VAL GLY GLU VAL ASP GLN ILE GTC GGT TGT GCG GTA TTG GGA AAC AGT GCCGCG TTA GTT GTT GGC GAG GTG GAC CAA ATC 7681 ARG LEU GLN TYR GLY ILE PHEARG ILE HIS GLN GLU VAL GLU PRO GLU LYS GLY SER GLU AGG CTG CAG TAC GGAATC TTT CGT ATT CAT CAG GAA GTC GAG CCG GAA AAA GGC TCT GAA 7741 ASN ALAVAL ILE THR VAL PRO ALA ASP LEU SER ALA GLU GLU ARG GLY ARG ILE GLN GLUAAC GCA GTT ATA ACC GTT CCC GCA GAC CTT TCA GCA GAG GAG CGA GGA CGG ATACAG GAA 7801 THR ALA LYS LYS ILE TYR LYS ALA LEU GLY CYS ARG GLY LEU ALAARG VAL ASP MET PHE ACG GCA AAA AAA ATA TAT AAA GCG CTC GGC TGT AGA GGTCTA GCC CGT GTG GAT ATG TTT 7861 LEU GLN ASP ASN GLY ARG ILE VAL LEU ASNGLU VAL ASN THR LEU PRO GLY PHE THR SER TTA CAA GAT AAC GGC CGC ATT GTACTG AAC GAA GTC AAT ACT CTG CCC GGT TTC ACG TCA 7921 TYR SER ARG TYR PROARG MET MET ALA ALA ALA GLY ILE ALA LEU PRO GLU LEU ILE ASP TAC AGT CGTTAT CCC CGT ATG ATG GCC GCT GCA GGT ATT GCA CTT CCC GAA CTG ATT GAC 7981ARG LEU ILE VAL LEU ALA LEU LYS GLY CGC TTG ATC GTA TTA GCG TTA AAG GGGTGATAAGC ATG GAA ATA GGA TTT ACT TTT TTA GAT                                    VanX    MET GLU ILE GLY PHE THRPHE  LEU ASP 8043 GLU ILE VAL HIS GLY VAL ARG TRP ASP ALA LYS TYR ALATHR TRP ASP ASN PHE THR GLY GAA ATA GTA CAC GGT GTT CGT TGG GAC GCT AAATAT GCC ACT TGG GAT AAT TTC ACC GGA 8103 LYS PRO VAL ASP GLY TYR GLU VALASN ARG ILE VAL GLY THR TYR GLU LEU ALA GLU SER AAA CCG GTT GAC GGT TATGAA GTA AAT CGC ATT GTA GGG ACA TAC GAG TTG GCT GAA TCG 8163 LEU LEU LYSALA LYS GLU LEU ALA ALA THR GLN GLY TYR GLY LEU LEU LEU TRP ASP GLY CTTTTG AAG GCA AAA GAA CTG GCT GCT ACC CAA GGG TAC GGA TTG CTT CTA TGG GACGGT 8223 TYR ARG PRO LYS ARG ALA VAL ASN CYS PHE MET GLN TRP ALA ALA GLNPRO GLU ASN ASN TAC CGT CCT AAG CGT GCT GTA AAC TGT TTT ATG CAA TGG GCTGCA CAG CCG GAA AAT AAC 8283 LEU THR LYS GLU SER TYR TYR PRO ASN ILE ASPARG THR GLU MET ILE SER LYS GLY TYR CTG ACA AAG GAA AGT TAT TAT CCC AATATT GAC CGA ACT GAG ATG ATT TCA AAA GGA TAC 8343 VAL ALA SER LYS SER SERHIS SER ARG GLY SER ALA ILE ASP LEU THR LEU TYR ARG LEU GTG GCT TCA AAATCA AGC CAT AGC CGC GGC AGT GCC ATT GAT CTT ACG CTT TAT CGA TTA 8403 ASPTHR GLY GLU LEU VAL PRO MET GLY SER ARG PHE ASP PHE MET ASP GLU ARG SERHIS GAC ACG GGT GAG CTT GTA CCA ATG GGG AGC CGA TTT GAT TTT ATG GAT GAACGC TCT CAT 8463 HIS ALA ALA ASN GLY ILE SER CYS ASN GLU ALA GLN ASN ARGARG ARG LEU ARG SER ILE CAT GCG GCA AAT GGA ATA TCA TGC AAT GAA GCG CAAAAT CGC AGA CGT TTG CGC TCC ATC 8523 MET GLU ASN SER GLY PHE GLU ALA TYRSER LEU GLU TRP TRP HIS TYR VAL LEU ARG ASP ATG GAA AAC AGT GGG TTT GAAGCA TAT AGC CTC GAA TGG TGG CAC TAT GTA TTA AGA GAC 8583 GLU PRO TYR PROASN SER TYR PHE ASP PHE PRO VAL LYS GAA CCA TAC CCC AAT AGC TAT TTT GATTTC CCC GTT AAA TAAA CTT TTA ACC GTT GCA 8641 CGG ACA AAC TAT ATA AGCTAA CTC TTT CGG CAG GAA ACC CGA CGT ATG TAA CTG GTT CTT 8701 AGG GAA TTTATA TAT AGT AGA TAG TAT TGA AGA TGT AAG GCA GAG CGA TAT TGC GGT CAT 8761TAT CTG CGT GCG CTG CGG CAA GAT AGC CTG ATA ATA AGA CTG ATC GCA TAG AGGGGT GGT 8821 ATT TCA CAC CGC CCA TTG TCA ACA GGC AGT TCA GCC TCG TTA AATTCA GCA TGG GTA TCA 8881 CTT ATG AAA ATT CAT CTA CAT TGG TGA TAA TAG TAAATC CAG TAG GGC GAA ATA ATT GAC 8941 TGT AAT TTA CGG GGC AAA ACG GCA CAATCT CAA ACG AGA TTG TGC CGT TTA AGG GGA AGA 9001                                                            VanY    METLYS LYS TTC TAG AAA TAT TTC ATA CTT CCA ACT ATA TAG TTA AGG AGG AGA CTGAAA ATG AAG AAG 9061 LEU PHE PHE LEU LEU LEU LEU LEU PHE LEU ILE TYR LEUGLY TYR ASP TYR VAL ASN GLU TTG TTT TTT TTA TTG TTA TTG TTA TTC TTA ATATAC TTA GGT TAT GAC TAC GTT AAT GAA 9121 ALA LEU PHE SER GLN GLU LYS VALGLU PHE GLN ASN TYR ASP GLN ASN PRO LYS GLU HIS GCA CTG TTT TCT CAG GAAAAA GTC GAA TTT CAA AAT TAT GAT CAA AAT CCC AAA GAA CAT 9181 LEU GLU ASNSER GLY THR SER GLU ASN THR GLN GLU LYS THR ILE THR GLU GLU GLN VAL TTAGAA AAT AGT GGG ACT TCT GAA AAT ACC CAA GAG AAA ACA ATT ACA GAA GAA CAGGTT 9241 TYR GLN GLY ASN LEU LEU LEU ILE ASN SER LYS TYR PRO VAL ARG GLNGLU SER VAL LYS TAT CAA GGA AAT CTG CTA TTA ATC AAT AGT AAA TAT CCT GTTCGC CAA GAA AGT GTG AAG 9301 SER ASP ILE VAL ASN LEU SER LYS HIS ASP GLULEU ILE ASN GLY TYR GLY LEU LEU ASP TCA GAT ATC GTG AAT TTA TCT AAA CATGAC GAA TTA ATA AAT GGA TAC GGG TTG CTT GAT 9361 SER ASN ILE TYR MET SERLYS GLU ILE ALA GLN LYS PHE SER GLU MET VAL ASN ASP ALA AGT AAT ATT TATATG TCA AAA GAA ATA GCA CAA AAA TTT TCA GAG ATG GTC AAT GAT GCT 9421 VALLYS GLY GLY VAL SER HIS PHE ILE ILE ASN SER GLY TYR ARG ASP PHE ASP GLUGLN GTA AAG GGT GGC GTT AGT CAT TTT ATT ATT AAT AGT GGC TAT CGA GAC TTTGAT GAG CAA 9481 SER VAL LEU TYR GLN GLU MET GLY ALA GLU TYR ALA LEU PROALA GLY TYR SER GLU HIS AGT GTG CTT TAC CAA GAA ATG GGG GCT GAG TAT GCCTTA CCA GCA GGT TAT AGT GAG CAT 9541 ASN SER GLY LEU SER LEU ASP VAL GLYSER SER LEU THR LYS MET GLU ARG ALA PRO GLU AAT TCA GGT TTA TCA CTA GATGTA GGA TCA AGC TTG ACG AAA ATG GAA CGA GCC CCT GAA 9601 GLY LYS TRP ILEGLU GLU ASN ALA TRP LYS TYR GLY PHE ILE LEU ARG TYR PRO GLU ASP GGA AAGTGG ATA GAA GAA AAT GCT TGG AAA TAC GGG TTC ATT TTA CGT TAT CCA GAG GAC9661 LYS THR GLU LEU THR GLY ILE GLN TYR GLU PRO TRP HIS ILE ARG TYR VALGLY LEU PRO AAA ACA GAG TTA ACA GGA ATT CAA TAT GAA CCA TGG CAT ATT CGCTAT GTT GGT TTA CCA 9721 HIS SER ALA ILE MET LYS GLU LYS ASN PHE VAL LEUGLU GLU TYR MET ASP TYR LEU LYS CAT AGT GCG ATT ATG AAA GAA AAG AAT TTCGTT CTC GAG GAA TAT ATG GAT TAC CTA AAA 9781 GLU GLU LYS THR ILE SER VALSER VAL ASN GLY GLU LYS TYR GLU ILE PHE TYR TYR PRO GAA GAA AAA ACC ATTTCT GTT AGT GTA AAT GGG GAA AAA TAT GAG ATC TTT TAT TAT CCT 9841 VAL THRLYS ASN THR THR ILE HIS VAL PRO THR ASN LEU ARG TYR GLU ILE SER GLY ASNGTT ACT AAA AAT ACC ACC ATT CAT GTG CCG ACT AAT CTT CGT TAT GAG ATA TCAGGA AAC 9901 ASN ILE ASP GLY VAL ILE VAL THR VAL PHE PRO GLY SER THR HISTHR ASN SER ARG ARG AAT ATA GAC GGT GTA ATT GTG ACA GTG TTT CCC GGA TCAACA CAT ACT AAT TCA AGG AGG 9961 TAA GGA TGG CGG AAT GAA ACC AAC GAA ATTAAT GAA CAG CAT TAT TGT ACT AGC ACT TTT 10021 GGG GTA ACG TTA GCT TTTTAA TTT AAA ACC CAC GTT AAC TAG GAC ATT GCT ATA CTA ATG 10081                                   VanZ     LEU GLY LYS ILE LEU SER  ARGGLY LEU ATA CAA CTT AAA CAA AAG AATTAGAGG AAA TTA TA TTG GGA AAA ATA TTATCT AGA GGA TTG 10143 LEU ALA LEU TYR LEU VAL THR LEU ILE TRP LEU VALLEU PHE LYS LEU GLN TYR ASN ILE CTA GCT TTA TAT TTA GTG ACA CTA ATC TGGTTA GTG TTA TTC AAA TTA CAA TAC AAT ATT 10203 LEU SER VAL PHE ASN TYRHIS GLN ARG SER LEU ASN LEU THR PRO PHE THR ALA THR GLY TTA TCA GTA TTTAAT TAT CAT CAA AGA AGT CTT AAC TTG ACT CCA TTT ACT GCT ACT GGG 10263ASN PHE ARG GLU MET ILE ASP ASN VAL ILE ILE PHE ILE PRO PHE GLY LEU LEULEU ASN AAT TTC AGA GAG ATG ATA GAT AAT GTT ATA ATC TTT ATT CCA TTT GGCTTG CTT TTG AAT 10323 VAL ASN PHE LYS GLU ILE GLY PHE LEU PRO LYS PHEALA PHE VAL LEU VAL LEU SER LEU GTC AAT TTT AAA GAA ATC GGA TTT TTA CCTAAG TTT GCT TTT GTA CTG GTT TTA AGT CTT 10383 THR PHE GLU ILE ILE GLNPHE ILE PHE ALA ILE GLY ALA THR ASP ILE THR ASP VAL ILE ACT TTT GAA ATAATT CAA TTT ATC TTC GCT ATT GGA GCG ACA GAC ATA ACA GAT GTA ATT 10443THR ASN THR VAL GLY GLY PHE LEU GLY LEU LYS LEU TYR GLY LEU SER ASN LYSHIS MET ACA AAT ACT GTT GGA GGC TTT CTT GGA CTG AAA TTA TAT GGT TTA AGCAAT AAG CAT ATG 10503 ASN GLN LYS LYS LEU ASP ARG VAL ILE ILE PHE VALGLY ILE LEU LEU LEU VAL LEU LEU AAT CAA AAA AAA TTA GAC AGA GTT ATT ATTTTT GTA GGT ATA CTT TTG CTC GTA TTA TTG 10563 LEU VAL TYR ARG THR HISLEU ARG ILE ASN TYR VAL CTC GTT TAC CGT ACC CAT TTA AGA ATA AAT TAC GTGTAAG ATG TCT AAA TCA AGC AAT 10621 CTG ATC TTT CAT ACA CAT AAA GAT ATTGAA TGA ATT GGA TTA GAT GGA AAA CGG GAT GTG 10681 GGG AAA CTC GCC CGTAGG TGT GAA GTG AGG GGA AAA CCG GTG ATA AAG TAA AAA GCT TAC 10741 CTAACA CTA TAG TAA CAA AGA AAG CCC AAT TAT CAA TTT TAG TGC TGA GGA ATT GGTCTC 10801 TTT AAT AAA TTT CCT TAA CGT TGT AAA TCC GCA TTT TCC TGA CGGTAC CCC Ib (−) Strand (corresponds to the sequence of the strandcomplementary to the (+) strand from 1 to 3189. 1 CAA AAT ATC ACC TCATTT TTG AGA CAA GTC TTA TGA GAC GCT CTT AAC TAT GAT TTT ATC 61 AGT CTACTA CAT TTG TAT CAA TAG AGT ACA CTC TAT TGA TAT ATA ATT GAA CTA ATA AAT121                Transposase          MET LYS ILE ALA ARG GLY ARGGLU  LEU LEU THR TGA AAA TAC AGA AAT GGA ATGATACTG AA ATG AAA ATT GCGAGA GGT AGA GAA TTG CTT ACA 182 PRO GLU GLN ARG GLN ALA PHE MET GLN ILEPRO GLU ASP GLU TRP ILE LEU GLY THR TYR CCG GAA CAG AGA CAG GCT TTT ATGCAA ATC CCT GAA GAT GAA TGG ATA CTG GGG ACC TAC 242 PHE THR PHE SER LYSARG ASP LEU GLU ILE VAL ASN LYS ARG ARG ARG GLU GLU ASN ARG TTC ACT TTTTCC AAA CGG GAT TTA GAA ATA GTT AAT AAG CGA AGG AGG GAA GAA AAC CGT 302LEU GLY PHE ALA VAL GLN LEU ALA VAL LEU ARG TYR PRO GLY TRP PRO TYR THRHIS ILE TTA GGA TTT GCT GTT CAA TTA GCT GTT CTT CGG TAT CCC GGT TGG CCATAC ACT CAT ATC 362 LYS SER ILE PRO ASP SER VAL ILE GLN TYR ILE SER LYSGLN ILE GLY VAL SER PRO SER AAA AGC ATC CCA GAT TCG GTC ATA CAA TAT ATATCG AAA CAG ATT GGT GTT AGT CCA TCC 422 SER LEU ASP HIS TYR PRO GLN ARGGLU ASN THR LEU TRP ASP HIS LEU LYS GLU ILE ARG TCG CTT GAT CAT TAT CCTCAA AGG GAA AAT ACA CTT TGG GAT CAT TTG AAA GAA ATT CGA 482 SER GLU TYRASP PHE VAL THR PHE THR LEU SER GLU TYR ARG MET THR PHE LYS TYR LEU AGTGAA TAC GAC TTT GTA ACT TTT ACC CTG AGT GAA TAT CGA ATG ACA TTT AAG TACCTT 542 HIS GLN LEU ALA LEU GLU ASN GLY ASP ALA ILE HIS LEU LEU HIS GLUCYS ILE ASP PHE CAT CAA TTA GCT TTG GAA AAT GGT GAT GCC ATT CAT CTA CTGCAT GAA TGC ATA GAT TTT 602 LEU ARG LYS ASN LYS ILE ILE LEU PRO ALA ILETHR THR LEU GLU ARG MET VAL TRP GLU CTA AGA AAA AAC AAA ATT ATA CTG CCTGCT ATC ACT ACA CTT GAA AGA ATG GTG TGG GAA 662 ALA ARG ALA MET ALA GLULYS LYS LEU PHE ASN THR VAL SER LYS SER LEU THR ASN GLU GCA AGG GCA ATGGCT GAA AAG AAG CTA TTT AAT ACG GTT AGT AAA TCT CTA ACA AAT GAG 722 GLNLYS GLU LYS LEU GLU GLY ILE ILE THR SER GLN HIS PRO SER GLU SER ASN LYSTHR CAA AAA GAA AAG CTT GAA GGG ATT ATT ACC TCG CAG CAT CCA TCC GAA TCCAAT AAA ACG 782 ILE LEU GLY TRP LEU LYS GLU PRO PRO GLY HIS PRO SER PROGLU THR PHE LEU LYS ILE ATA TTG GGT TGG TTA AAA GAG CCA CCG GGT CAT CCTTCA CCC GAA ACT TTT CTA AAA ATA 842 ILE GLU ARG LEU GLU TYR ILE ARG GLYMET ASP LEU GLU THR VAL GLN ILE SER HIS LEU ATA GAA CGA CTC GAA TAC ATACGA GGA ATG GAT TTA GAA ACA GTG CAA ATT AGT CAT TTG 902 HIS ARG ASN ARGLEU LEU GLN LEU SER ARG LEU GLY SER ARG TYR GLU PRO TYR ALA PHE CAC CGTAAC CGC CTG TTG CAG CTG TCT CGC TTA GGC TCA AGA TAC GAG CCG TAT GCA TTC962 ARG ASP PHE GLN GLU ASN LYS ARG TYR SER ILE LEU THR ILE TYR LEU LEUGLN LEU THR CGT GAC TTT CAA GAA AAT AAA CGT TAT TCG ATA TTA ACC ATC TATTTA TTA CAA CTT ACT 1022 GLN GLU LEU THR ASP LYS ALA PHE GLU ILE HIS ASPARG GLN ILE LEU SER LEU LEU SER CAG GAG CTA ACG GAT AAA GCG TTT GAA ATTCAT GAT AGG CAA ATA CTT AGT TTG TTA TCA 1082 LYS GLY ARG LYS ALA GLN GLUGLU ILE GLN LYS GLN ASN GLY LYS LYS LEU ASN GLU LYS AAA GGT CGT AAG GCTCAA GAG GAA ATC CAG AAA CAA AAC GGT AAA AAG CTA AAT GAG AAA 1142 VAL ILEHIS PHE THR ASN ILE GLY GLN ALA LEU ILE LYS ALA ARG GLU GLU LYS LEU ASPGTT ATA CAC TTT ACG AAC ATC GGA CAA GCA TTA ATT AAA GCA AGA GAG GAA AAATTA GAC 1202 VAL PHE LYS VAL LEU GLU SER VAL ILE GLU TRP ASN THR PHE VALSER SER VAL GLU GLU GTT TTT AAG GTT TTA GAA TCG GTT ATT GAA TGG AAT ACCTTT GTC TCT TCA GTA GAA GAG 1262 ALA GLN GLU LEU ALA ARG PRO ALA ASP TYRASP TYR LEU ASP LEU LEU GLN LYS ARG PHE GCT CAG GAA CTT GCA CGT CCT GCCGAC TAT GAT TAT TTA GAC TTA CTG CAA AAA CGG TTT 1322 TYR SER LEU ARG LYSTYR THR PRO THR LEU LEU ARG VAL LEU GLU PHE HIS SER THR LYS TAT TCA CTAAGA AAA TAT ACG CCA ACG CTA TTA AGA GTA TTG GAA TTT CAT TCT ACA AAG 1382ALA ASN GLU PRO LEU LEU GLN ALA VAL GLU ILE ILE ARG GLY MET ASN GLU SERGLY LYS GCA AAT GAG CCA CTT TTA CAA GCT GTT GAG ATT ATC CGA GGA ATG AACGAA TCT GGA AAG 1442 ARG LYS VAL PRO ASP ASP SER PRO VAL ASP PHE ILE SERLYS ARG TRP LYS ARG HIS LEU CGA AAA GTG CCT GAT GAC TCA CCT GTG GAT TTTATT TCA AAA CGA TGG AAA AGA CAT TTA 1502 TYR GLU ASP ASP GLY THR THR ILEASN ARG HIS TYR TYR GLU MET ALA VAL LEU THR GLU TAC GAG GAT GAT GGT ACAACA ATT AAT CGT CAT TAC TAT GAA ATG GCT GTT TTA ACA GAA 1562 LEU ARG GLUHIS VAL ARG ALA GLY ASP VAL SER ILE VAL GLY SER ARG GLN TYR ARG ASP CTTCGG GAG CAT GTT CGG GCA GGA GAT GTT TCC ATT GTT GGC AGC AGA CAA TAT AGGGAT 1622 PHE GLU GLU TYR LEU PHE SER GLU ASP THR TRP ASN GLN SER LYS GLYASN THR ARG LEU TTT GAG GAA TAT TTG TTT TCG GAA GAT ACA TGG AAT CAA TCGAAG GGG AAT ACG AGA TTA 1682 SER VAL SER LEU SER PHE GLU ASP TYR ILE THRGLU ARG THR SER SER PHE ASN GLU ARG TCA GTT AGT TTA TCA TTC GAA GAT TATATA ACG GAG AGA ACC AGC AGC TTT AAT GAA AGG 1742 LEU LYS TRP LEU ALA ALAASN SER ASN LYS LEU ASP GLY VAL SER LEU GLU LYS GLY LYS TTA AAG TGG TTAGCT GCC AAT TCC AAT AAG TTA GAT GGG GTT TCT CTT GAA AAA GGA AAG 1802 LEUSER LEU ALA ARG LEU GLU LYS ASP VAL PRO GLU GLU ALA LYS LYS PHE SER ALASER CTA TCA CTT GCA CGC TTA GAA AAA GAT GTT CCA GAA GAA GCA AAA AAA TTTAGT GCA AGC 1862 LEU TYR GLN MET LEU PRO ARG ILE LYS LEU THR ASP LEU LEUMET ASP VAL ALA HIS ILE CTT TAT CAG ATG CTA CCA AGA ATA AAA TTA ACT GATTTA CTC ATG GAT GTG GCC CAT ATA 1922 THR GLY PHE HIS GLU GLN PHE THR HISALA SER ASN ASN ARG LYS PRO ASP LYS GLU GLU ACA GGA TTT CAT GAG CAA TTCACT CAT GCT TCC AAT AAT CGA AAA CCA GAT AAG GAA GAA 1982 THR ILE ILE ILEMET ALA ALA LEU LEU GLY MET GLY MET ASN ILE GLY LEU SER LYS MET ACA ATCATT ATC ATG GCT GCC CTT TTA GGA ATG GGA ATG AAT ATT GGC TTG AGC AAG ATG2042 ALA GLU ALA THR PRO GLY LEU THR TYR LYS GLN LEU ALA ASN VAL SER GLNTRP ARG MET GCC GAA GCC ACA CCC GGA CTT ACA TAT AAG CAA CTA GCC AAT GTATCT CAA TGG CGC ATG 2102 TYR GLU ASP ALA MET ASN LYS ALA GLN ALA ILE LEUVAL ASN PHE HIS HIS LYS LEU GLN TAT GAA GAT GCC ATG AAT AAA GCC CAA GCCATA TTA GTA AAC TTT CAT CAT AAA TTA CAA 2162 LEU PRO PHE TYR TRP GLY ASPGLY THR THR SER SER SER ASP GLY MET ARG MET GLN LEU TTG CCT TTC TAT TGGGGC GAC GGT ACA ACA TCT TCG TCA GAT GGT ATG AGA ATG CAG CTA 2222 GLY VALSER SER LEU HIS ALA ASP ALA ASN PRO HIS TYR GLY THR GLY LYS GLY ALA THRGGT GTT TCA TCA CTA CAT GCA GAT GCA AAT CCA CAT TAT GGA ACT GGA AAA GGAGCC ACC 2282 ILE TYR ARG PHE THR SER ASP GLN PHE SER SER TYR TYR THR LYSILE ILE HIS THR ASN ATC TAC CGA TTT ACA AGT GAT CAA TTC TCT TCT TAC TACACA AAG ATT ATT CAT ACT AAT 2342 SER ARG ASP ALA ILE HIS VAL LEU ASP GLYLEU LEU HIS HIS GLU THR ASP LEU ASN ILE TCA AGA GAT GCG ATT CAT GTT TTGGAT GGT TTG TTA CAT CAT GAG ACG GAT CTA AAC ATA 2402 GLU GLU HIS TYR THRASP THR ALA GLY TYR THR ASP GLN ILE PHE GLY LEU THR HIS LEU GAG GAA CATTAT ACA GAC ACT GCC GGT TAC ACT GAC CAA ATA TTC GGA CTG ACT CAT TTA 2462LEU GLY PHE LYS PHE ALA PRO ARG ILE ARG ASP LEU SER ASP SER LYS LEU PHETHR ILE TTA GGA TTT AAA TTT GCC CCA AGA ATA AGG GAT TTA TCG GAC TCA AAATTA TTT ACG ATA 2522 ASP LYS ALA SER GLU TYR PRO LYS LEU GLU ALA ILE LEUARG GLY GLN ILE ASN THR LYS GAT AAA GCA AGT GAG TAT CCA AAA CTA GAA GCCATT TTA CGT GGA CAA ATA AAT ACA AAG 2582 VAL ILE LYS GLU ASN TYR GLU ASPVAL LEU ARG LEU ALA HIS SER ILE ARG GLU GLY THR GTC ATT AAA GAA AAT TATGAG GAT GTT TTG CGA TTA GCT CAT TCT ATA AGG GAG GGA ACA 2642 AGT TTC AGCATC CCT TAT TAT GGG GAA GCT AGG TTC CTA TTC AAG ACA AAA CAG CTT AGC VALSER ALA SER LEU ILE MET GLY LYS LEU GLY SER TYR SER ARG GLN ASN SER LEUALA GTT TCA GCA TCC CTT ATT ATG GGG AAG CTA GGT TCC TAT TCA AGA CAA AACAGC TTA GCT 2702 THR ALA LEU ARG GLU MET GLY ARG ILE GLU LYS THR ILE PHEILE LEU ASN TYR ILE SER ACA GCC TTA CGT GAG ATG GGC CGA ATA GAA AAA ACGATC TTT ATT TTG AAT TAT ATA TCG 2762 ASP GLU SER LEU ARG ARG LYS ILE GLNARG GLY LEU ASN LYS GLY GLU ALA MET ASN GLY GAT GAA TCA TTA AGA AGA AAAATA CAA AGA GGA TTG AAT AAA GGA GAA GCC ATG AAT GGA 2822 LEU ALA ARG ALAILE PHE PHE GLY LYS GLN GLY GLU LEU ARG GLU ARG THR ILE GLN HIS TTG GCAAGA GCT ATT TTC TTC GGA AAA CAA GGT GAG CTT AGA GAA CGC ACC ATA CAG CAT2882 GLN LEU GLN ARG ALA SER ALA LEU ASN ILE ILE ILE ASN ALA ILE SER ILETRP ASN THR CAA TTG CAA AGA GCC AGT GCT TTA AAC ATA ATT ATC AAT GCT ATAAGT ATT TGG AAT ACT 2942 TCT CCA CCT AAC AAC AGC AGT TGA ATA TAA AAA ACGGAC AGG TAG CTT TAA TGA AGA TTT LEU HIS LEU THR THR ALA VAL GLU TYR LYSLYS ARG THR GLY SER PHE ASN GLU ASP LEU CTC CAC CTA ACA ACA GCA GTT GAATAT AAA AAA CGG ACA GGT AGC TTT AAT GAA GAT TTG 3002 LEU HIS HIS MET SERPRO LEU GLY TRP GLU HIS ILE ASN LEU LEU GLY GLU TYR HIS PHE TTA CAC CATATG TCG CCC TTA GGT TGG GAA CAT ATT AAT TTA CTA GGA GAA TAC CAT TTT 3062ASN SER GLU LYS VAL VAL SER LEU ASN SER LEU ARG PRO LEU LYS LEU SER AACTCA GAG AAA GTA GTC TCA TTA AAT TCT TTA AGA CCA CTA AAA CTT TCT TAA CGTTG 3121 TTA AAA ACG AGG GAT TCG TCA GGA AAA TAG GCT TAG CGT TGT AAA TCCGCA TTT TCC TGA 3181 CGC TAC CCC LIST OF SEQUENCES : ii     SacI 42                           GAGCTCTTCCTTCAACGCACTTCTGTACCAAGAGTTGTTGTCCATTTGATCACTAACAATAGCTTCCCCTGCTTTCTTCAAGCCCTTTGTCATAAAATCGTTAGATTTTCA111TCATAAAAATACGAGAAAGACAACAGGAAGACCGCAAATTTTCTTTTCTTTTCCTAGGTACACTGAATG180                       RBS          M  K  K  I  A  V  L  F  G  G 244TAACCTTAAAAGAAAAAAGGAAAGGAAGAAAATGATGAAAAAAATTGCCGTTTTATTTGGAGGG N  S  P  E  Y  S  V  S  L  T  S  A  A  S  V  I  Q  A  I  D 304AATTCTCCAGAATACTCAGTGTCACTAACCTCAGCAGCAAGTGTGATCCAAGCTATTGAC P  L  K  Y  E  V  N  T  I  G  I  A  P  T  M  D  W  Y  W  Y 364CCGCTGAAATATGAAGTAATGACCATTGGCATCGCACCAACAATGGATTGGTATTGGTAT Q  G  M  L  A  N  V  R  N  D  T  W  L  E  D  H  K  N  C  H 424CAAGGAAACCTCGCGAATGTTCGCAATGATACTTGGCTAGAAGATCACAAAAACTGTCAC Q  L  T  F  S  S  Q  G  F  I  L  G  E  K  R  I  V  P  D  V 484CAGCTGACTTTTTCTAGCCAAGGATTTATATTAGGAGAAAAACGAATCGTCCCTGATGTC L  F  P  V  L  H  G  K  Y  G  E  D  G  C  I  Q  G  L  L  E 544CTCTTTCCAGTCTTGCATGGGAAGTATGGCGAGGATGGCTGTATCCAAGGACTGCTTGAA L  M  N  L  P  Y  V  G  C  H  V  A  A  S  A  L  C  M  N  K 604CTAATGAACCTGCCTTATGTTGGTTGCCATGTCGCTGCCTCCGCATTATGTATGAACAAA W  L  L  H  Q  L  A  D  T  M  G  I  A  S  A  P  T  L  L  L 664TGGCTCTTGCATCAACTTGCTGATACCATGGGAATCGCTAGTGCTCCCACTTTGCTTTTA S  R  Y  E  N  D  P  A  T  I  D  R  F  I  Q  D  H  G  F  P 724TCCCGCTATGAAAACGATCCTGCCACAATCGATCGTTTTATTCAAGACCATGGATTCCCG I  F  I  K  P  N  E  A  G  S  S  K  G  I  T  K  V  T  D  K 784ATCTTTATCAAGCCGAATGAAGCCGGTTCTTCAAAAGGGATCACAAAAGTAACTGACAAA T  A  L  Q  S  A  L  T  T  A  F  A  Y  G  S  T  V  L  I  Q 844ACAGCGCTCCAATCTGCATTAACGACTGCTTTTGCTTACGGTTCTACTGTGTTGATCCAA K  A  I  A  G  I  E  I  G  C  G  I  L  G  N  E  Q  L  T  I 904AAGGCGATAGCGGGTATTGAAATTGGCTGCGGCATCTTAGGAAATGAGCAATTGACGATT G  A  C  D  A  I  S  L  V  D  G  F  F  D  F  E  E  K  Y  Q 964GGTGCTTGTGATGCGATTTCTCTTGTCGACGGTTTTTTTGATTTTGAAGAGAAATACCAA L  I  S  A  T  I  T  V  P  A  P  L  P  L  A  L  E  S  Q  I 1024TTAATCAGCGCCACGATCACTGTCCCAGCACCATTGCCTCTCGCGCTTGAATCACAGATC K  E  Q  A  Q  L  L  Y  R  N  L  G  L  T  G  L  A  R  I  D 1084AAGGAGCAGGCACAGCTGCTTTATCGAAACTTGGGATTGACGGGTCTGGCTCGAATCGAT F  F  V  T  N  Q  G  A  I  Y  L  N  E  I  N  T  M  P  G  F 1144TTTTTCGTCACCAATCAAGGAGCGATTTATTTAAACGAAATCAACACCATGCCGGGATTT T  G  H  S  R  Y  P  A  M  M  A  E  V  G  L  S  Y  E  I  L 1204ACTGGGCACTCCCGCTACCCAGCTATGATGGCGGAAGTCGGGTTATCCTACGAAATATTA V  E  Q  L  E  A  L  A  E  E  D  K  R  * 1267GTAGAGCAATTGATTGCACTGGCAGAGGAGGACAAACGATGAACACATTACAATTGATCAATAAAAACCATCCATTGAAAAAAAATCAAGAGCCCCCGCACTTAGTGCTAGCTCCTTTTAGCGATCACGATG1336 TTTACCTGCAG 1347        PstI

1. A composition comprising: (a) an isolated protein having the aminoacid sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, wherein saidprotein or fragment when combined with (b) and (c) confers resistance toglycopeptides in Gram-positive bacteria; (b) an isolated protein havingthe amino acid sequence of SEQ ID NO:6 or a fragment of SEQ ID NO:6,wherein said protein or fragment when combined with (a) and (c) confersresistance to glycopeptides in Gram-positive bacteria; and (c) anisolated protein or protein fragment selected from the group consistingof a protein having the amino acid sequence of SEQ ID NO:4, a fragmentof SEQ ID NO:4, a protein having the amino acid sequence of SEQ IDNO:25, and a fragment of SEQ ID NO:25, wherein said protein or proteinfragment when combined with (a) and (b) confers resistance toglycopeptides in Gram-positive bacteria; wherein the composition confersresistance to glycopeptides in Gram-positive bacteria.
 2. Thecomposition of claim 1, which comprises the isolated protein having theamino acid sequence of SEQ ID NO:2, the isolated protein having theamino acid sequence of SEQ ID NO:6, and the isolated protein having theamino acid sequence of SEQ ID NO:4.
 3. The composition of claim 1, whichcomprises the isolated protein having the amino acid sequence of SEQ IDNO:2, the isolated protein having the amino acid sequence of SEQ IDNO:6, and the isolated protein having the amino acid sequence of SEQ IDNO:25.
 4. A composition comprising: (a) an isolated protein encoded by anucleotide sequence that hybridizes to SEQ ID NO: 17, or a proteinencoded by a nucleotide sequence that hybridizes to SEQ ID NO:3, whereinsaid protein when combined with (b) and (c) confers resistance toglycopeptides in Gram-positive bacteria; (b) an isolated protein encodedby a nucleotide sequence that hybridizes to SEQ ID NO:1, wherein saidprotein when combined with (a) and (c) confers resistance toglycopeptides in Gram-positive bacteria; and (c) an isolated proteinencoded by a nucleotide sequence that hybridizes to SEQ ID NO:5, whereinsaid protein when combined with (a) and (b) confers resistance toglycopeptides in Gram-positive bacteria; wherein the hybridizationconditions are under high stringency conditions, wherein the highstringency conditions comprise hybridization overnight at 65° C. in asolution containing 0.1% SDS, 0.7% skim milk powder, 6×SSC and washingat 65° C. in 2×SSC, and 0.1% SDS.
 5. The composition of claim 4, whichcomprises the isolated protein encoded by the nucleotide sequence thathybridizes to SEQ ID NO:17, the isolated protein encoded by thenucleotide sequence that hybridizes to SEQ ID NO:1; and the isolatedprotein encoded by the nucleotide sequence that hybridizes to SEQ IDNO:5.
 6. The composition of claim 4, which comprises the isolatedprotein encoded by the nucleotide sequence that hybridizes to SEQ IDNO:3, the isolated protein encoded by the nucleotide sequence thathybridizes to SEQ ID NO:1; and the isolated protein encoded by thenucleotide sequence that hybridizes to SEQ ID NO:5.